Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications.

Biotechnology Advances 32 (2014) 415–428

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Recent advances in discovery, heterologous expression, and molecular engineering of cyclodextrin glycosyltransferase for versatile applications Ruizhi Han a,b,c, Jianghua Li a,b,c, Hyun-dong Shin d, Rachel R. Chen d, Guocheng Du a,b,c,⁎, Long Liu a,b,c,⁎⁎, Jian Chen e a

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China d School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, USA e National Engineering of Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China b c

a r t i c l e

i n f o

Article history: Received 30 June 2013 Received in revised form 4 December 2013 Accepted 16 December 2013 Available online 20 December 2013 Keywords: Cyclodextrin glycosyltransferase (CGTase) Heterologous expression Molecular modification Directed evolution

a b s t r a c t Cyclodextrin glycosyltransferase (CGTase) is an important enzyme with multiple functions, in particular the production of cyclodextrins. It is also widely applied in baking and carbohydrate glycosylation because it participates in various types of catalytic reactions. New applications are being found with novel CGTases being isolated from various organisms. Heterologous expression is performed for the overproduction of CGTases to meet the requirements of these applications. In addition, various directed evolution techniques have been applied to modify the molecular structure of CGTase for improved performance in industrial applications. In recent years, substantial progress has been made in the heterologous expression and molecular engineering of CGTases. In this review, we systematically summarize the heterologous expression strategies used for enhancing the production of CGTases. We also outline and discuss the molecular engineering approaches used to improve the production, secretion, and properties (e.g., product and substrate specificity, catalytic efficiency, and thermal stability) of CGTase. © 2013 Elsevier Inc. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources, properties, and applications of CGTases . . . . . . . . . . . . . . . . . . . . . 2.1. Novel CGTases from various sources . . . . . . . . . . . . . . . . . . . . . . . 2.2. Applications of CGTases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heterologous expression of CGTase . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Heterologous expression for overproduction . . . . . . . . . . . . . . . . . . . 3.1.1. Molecular modification to improve CGTase secretion . . . . . . . . . . . 3.1.2. Molecular modification to improve CGTase overexpression . . . . . . . . 3.1.3. Cultivation strategy optimization to improve overproduction of CGTase. . . 3.2. Heterologous expression to improve properties . . . . . . . . . . . . . . . . . . Molecular engineering of CGTase to improve properties . . . . . . . . . . . . . . . . . 4.1. Site-directed mutagenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Site-directed mutagenesis for improving the product specificity of CGTases . 4.1.2. Site-directed mutagenesis for improving the substrate specificity of CGTases 4.1.3. Site-directed mutagenesis for improving the hydrolysis activity of CGTases . 4.1.4. Site-directed mutagenesis for improving the thermostability of CGTases . . . 4.2. Random mutation (error-prone polymerase chain reaction, epPCR) . . . . . . . . . 4.3. Fragment deletion mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Domain chimera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Molecular imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Correspondence to: G. Du, Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Tel./fax: +86 510 85918309. ⁎⁎ Correspondence to: L. Liu, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85918312; fax: +86 510 85918309. E-mail addresses: [email protected] (G. Du), [email protected] (L. Liu). 0734-9750/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biotechadv.2013.12.004

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5. Conclusions and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Cyclodextrin (CD) glucanotransferase (CGTase) (EC 2.4.1.19), also known as cyclodextrin glycosyltransferase, cyclomaltodextrin glycanotransferase, and cyclomaltodextrin glycosyltransferase, is a member of the α-amylase family of glycosyl hydrolases (family 13). Like the other members of this family, CGTase can catalyze hydrolysis reactions, cleaving the linkages in the starch molecule (which is followed by the reaction of the cleavage product with water, resulting in a new reducing end) (van der Veen et al., 2000a). However, CGTase usually has minor hydrolysis activity and mainly catalyzes three transglycosylation reactions— cyclization (cleavage of an α-glycosidic bond in amylose or starch and subsequent formation of a CD), coupling (cleavage of an α-glycosidic bond of a CD ring and transfer of the resulting maltooligosaccharide to a acceptor substrate), and disproportionation (cleavage of an α-glycosidic bond of a linear maltooligosaccharide and transfer of one part to an acceptor substrate) (van der Veen et al., 2000d). Among these reactions, cyclization is the specific reaction of CGTase and is often used for CD production; the coupling is considered the reverse reaction of the cyclization. More thorough understanding of the structure and mechanism of CGTases occurred in 1991 when the first structural model of the CGTase from Bacillus circulans was obtained via multiple isomorphous replacement and subsequent solvent-flattening at 2.5 Å resolution and refining at 2.0 Å resolution with a simulated annealing refinement method (Klein and Schulz, 1991). Subsequently, much attention has been paid to the 3-dimensional (3-D) structures of various CGTases. During the past 2 decades, 51 different CGTase crystal structures have been published; detailed information on these structures is listed in Table 1. These 3-D structures provide a basis for further research on mechanism and direct molecular engineering of CGTases. All of the crystallized CGTases have been isolated from bacteria. Specifically, 33 CGTases are from B. circulans (24 CGTases belong to B. circulans strain 251, and 9 belong to B. circulans strain 8), 11 are from Bacillus sp., 4 are from Thermoanaerobacterium thermosulfurigenes, and 3 are from Geobacillus stearothermophilus. The 3-D structures of CGTases from these sources are quite similar (N60%), as shown in Fig. 1 (PDB ID: 1CXK; Uitdehaag et al., 1999b), each with a 5-domain organization: A, B, C, D, and E. Domain A is a conventional (β/α)8 or triosephosphate isomerasebarrel structure containing a highly symmetrical fold of 8 parallel βstrands arranged in a barrel encircled by 8 α-helices (Janeček, 1994). This domain is present in all enzymes of the α-amylase family and is considered a catalytic area (van der Veen et al., 2000a). Domain B is a protuberant loop between β-strand 3 and α-helix 3 of domain A and contains 44 ~ 133 amino acid residues that play central roles in substrate binding (van der Veen et al., 2000a). Goh et al. (2012a) have reported a calcium-binding site at the A/B domain interface that could significantly influence the stability of CGTase by introducing a new salt bridge at the protein surface in domain B. Similarly, Leemhuis et al. (2004) improved the thermostability of B. circulans CGTase by replacing amino acid residues in the B domain and introducing a salt bridge. Goh et al. (2012b) further confirmed that the B domain of CGTase influences the thermostability of the enzyme via the exchange of B domains between different CGTases. Domains C, D, and E consist of the C-terminal region of CGTases. Domain C has 1 maltose-binding site and contributes to substrate binding (Penninga et al., 1996). Domain E has 2 maltose-binding sites and is regarded as a raw starch-binding domain (Dalmia et al., 1995; Penninga et al., 1996). Domain D is found exclusively in CGTases and its function remains unclear. The X-ray crystallographic structures of CGTases complexed with inhibitors, substrates, or products provide additional insights into the

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catalytic mechanisms of CGTase (see Table 1). CGTase has 3 active site residues: Glu257 is both a proton donor and acceptor, Asp229 forms a covalent intermediate with the cleaved substrate before CD formation, and Asp328 stabilizes the reaction intermediates (Uitdehaag et al., 1999b). The detailed catalytic mechanisms of CGTases have been described by van der Veen et al. (2000a) and are not discussed herein. At least 9 subsites, + 2 to − 7, for substrate binding are present around the active sites (Leemhuis et al., 2010). The +1 and +2 subsites stabilize the glucose ring with phenyl rings (Tachibana et al., 2000; Uitdehaag et al., 1999b), and the − 1 subsite contains the catalyzing center of CGTase (Kumar, 2010). Residues at the − 2 and −3 subsites have important effects on the 4 reactions (cyclization, coupling, disproportion, and hydrolysis) of CGTase (Han et al., 2013c; Li et al., 2009b,c; Liu et al., 2013; Yoon and Robyt, 2006). The −6 and −7 subsites bind the substrate (Leemhuis et al., 2002). Few studies have reported the functions of the −4 and −5 subsites, whereas the absence of strong interactions at the –4/–5 subsites has been indicated by molecular modeling studies to favor the selection of substrates long enough to form CDs (Uitdehaag et al., 2001). As the structure and mechanisms of CGTases have been clarified, the industrial application of CGTases has attracted interest. However, overproduction and enhancement of the properties of CGTases have become increasingly necessary to improve the quantity and quality of CGTases for efficient application. Previous CGTase reviews have focused mainly on yield (Schmid, 1989), catalytic mechanism and product specificity (van der Veen et al., 2000a), the CGTase gene and CGTase applications (Qi and Zimmermann, 2005), biotechnological applications (Leemhuis et al., 2010), and extracellular secretion strategies in Escherichia coli (Tesfai et al., 2012). In this review, we first introduce CGTases from various organisms and discuss their novel properties and applications. Then, we systematically summarize recent advances in heterologous expression strategies for improving CGTase production and molecular engineering approaches for enhancing the catalytic properties of CGTases for effective application. Finally, we discuss future prospects for CGTase-related researches. 2. Sources, properties, and applications of CGTases 2.1. Novel CGTases from various sources The first CGTase was obtained from Bacillus macerans in 1974 (Okada, 1974), and the first CGT gene in B. macerans was identified by Takano et al. (1986). Subsequently, additional CGTases were obtained from various sources, especially bacteria such as Bacillus, Paenibacillus, Klebsiella, Thermoanaerobacterium, and Thermoanaerobacter (Zheng et al., 2002). In addition, Abelian et al. (1995) have produced CGTase from Actinomycetes species. The main organism categories of native CGTases producers have been summarized in previous reviews (Qi and Zimmermann, 2005; Schmid, 1989). And some additional novel CGTases have recently been obtained from different sources. For instance, Lee et al. (2007) found a novel CGTase gene in Pyrococcus furiosus and expressed in Escherichia coli. The enzyme is extremely thermostable with an optimal temperature and pH of 95 °C and 5.0, respectively. A new β-CGTase with high specificity for the intermolecular transglycosylation of bioflavonoids excreted from alkalophilic Bacillus sp. BL-31 was obtained by Go et al. (2007). A novel thermostable alkaline CGTase was secreted from Bacillus pseudalcaliphilus strain 8SB (Atanasova et al., 2008), which converted starch into β-CD (CD7) and γ-CD (CD8) and had high production efficiency (Kitayska et al., 2011). Petrova et al. (2012) also isolated and


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Table 1 The three-dimensional structure of CGTases from different sources. Organism source

PDB ID

Resolution

Properties

Citation

Bacillus circulans strain 251

1PJ9 1PEZ 1OT1 1OT2 1KCK 1KCL 1EO5 1E07 1DTU 1D3C 1CXK 1CXL 2DIJ 2CXG 1TCM 1CXE 1CXF 1CXH 1CXI 1CGV 1CGW 1CGX 1CGY 1CDG 6CGT 8CGT 9CGT

2.00 Å 2.32 Å 2.00 Å 2.10 Å 2.43 Å 1.94 Å 2.00 Å 2.48 Å 2.40 Å 1.78 Å 2.09 Å 1.81 Å 2.60 Å 2.50 Å 2.10 Å 2.10 Å

Loop mutant 183–195 Mutant A230V Mutant D135A Mutant D135N Mutant N193G Mutant G179L Complex with maltoheptaose Complex with maltohexaose Mutant Y89D/S146P complex with an hexasaccharide inhibitor Complex with gamma-cyclodextrin Mutant E257Q/D229N complex with a maltononaose Mutant E257Q complex with a covalent intermediate Mutant Y195F complex with acarbose and maltohexaose Complex with acarbose Mutant W616A Complex with alpha-cyclodextrin Mutant D229N/E257Q complex with alpha-cyclodextrin Complex with maltotetraose and maltoheptaose 120 K and pH7.55 Mutant of Y195

Leemhuis et al. (2004) Leemhuis et al. (2003b) Leemhuis et al. (2003a) Leemhuis et al. (2003a) Leemhuis et al. (2002) Leemhuis et al. (2002) Uitdehaag et al. (2000) Uitdehaag et al. (2000) van der Veen et al. (2000c) Uitdehaag et al. (1999a) Uitdehaag et al. (1999b) Uitdehaag et al. (1999b) Strokopytov et al. (1996) Strokopytov et al. (1995) Penninga et al. (1996) Knegtel et al. (1995)

A maltose-dependent crystal form Complex with hoxa Complex with a thio-maltopentaose

Lawson et al. (1994) Parsiegla et al. (1998) Parsiegla et al. (1998)

Bacillus circulans strain 8

Bacillus sp.

Thermoanaerobacterium thermosulfurigenes

Geobacillus stearothermophillus

4CGT 5CGT 7CGT 3CGT 1CGT 1CGU 1V3J 1V3K 1V3L 1V3M 1UKQ 1UKS 1UKT 1DED 1D7F 1PAM 1I75 3BMV 3BMW 1A47 1CTU 1CYG 1QHO 1QHP

2.41 Å 2.20 Å 2.50 Å

2.00 Å 2.60 Å 2.40 Å 2.50 Å 2.60 Å 2.50 Å 3.00 Å 2.40 Å 2.00 Å 2.50 Å 2.00 Å 2.10 Å 2.00 Å 2.00 Å 1.90 Å 2.20 Å 2.00 Å 1.90 Å 1.80 Å 2.00 Å 1.60 Å 1.60 Å 2.56 Å 2.30 Å 2.50 Å 1.70 Å 1.70 Å

Deletion 145–150 and mutant F151D Complex with maltotriose Complex with RAMEB Complex with beta-cyclodextrin Wild-type Mutagenesis of catalytic center Mutant F283L Mutant F283Y Mutant F283L complex with a pseudo-tetraose Mutant F283Y complex with a pseudo-tetraose complex with a pseudo-tetraose Mutant F183L/F258L complex with a pseudo-maltotetraose Mutant Y100L complex with an acarbose Alkalophilic asparagine 233-replaced Wild-type Complex with 1-deoxynojirimycin Mutant S77P Mutant S77P complex with a maltoheptaose inhibitor complex with a maltoheptaose inhibitor Thermostabilization at pH 8.0 Wild-type maltose/acarbose complex maltose complex

sequenced a new cgt gene from the alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB, which had low level of homology with nucleotide sequences of other Bacillus CGTases (less than 82%). The first halophilic archaeal CGTase was isolated by Bautista et al. (2012) from the halophilic archaeon Haloferax mediterranei. Yenpetch et al. (2011) found a new CGTase in Paenibacillus sp. RB01, and its recombinant enzyme exhibited 3 isoforms (I, II, and III) with the same apparent size but different charges, demonstrating that the deamidation of labile Asn residues changes the molecular forms of CGTases. Ibrahim et al. (2012) obtained a novel CGTase a wide pH range of 5.0–11.0 from alkaliphilic Amphibacillus sp. NPST-10 isolated from hypersaline soda lakes (Wadi Natrun Valley, Egypt).

Penninga et al. (1995)

Schmidt et al. (1998) Klein and Schulz (1991) Klein et al. (1992) Kanai et al. (2004)

Haga et al. (2003)

Ishii et al. (2000) Harata et al. (1996) Kanai et al. (2001) Kelly et al. (2008) Wind et al. (1998) Knegtel et al. (1996) ND Dauter et al. (1999) Dauter et al. (1999)

production of CDs. According to the different CD specificities (α-CD [CD6], β-CD [CD7], or γ-CD [CD8]), CGTases are usually classified into 3 subgroups (α-, β-, and γ-CGTases), which often have different CD specificities. Table 2 shows the host organisms that produce various subgroup CGTases. Although CGTases are well known for their use in CD production, many additional applications have been explored as well. For example, CGTases are often used as antistaling agents in baked goods (Shim et al., 2004, 2007). Notably, CGTases also play an important role in improving carbohydrate properties such as solubility and stability via transglycosylation, alleviating bitterness, improving bifidogenic characteristics, lowering cytotoxicity, and extending shelf life (see Table 2).

2.2. Applications of CGTases 3. Heterologous expression of CGTase As a biocatalyst, CGTase has numerous applications related to its 4 catalytic reactions. Previous review (Qi and Zimmermann, 2005) has reviewed the applications of CGTases. The main application is the

Demand for CGTase is enormous owing to its central role in a variety of applications, especially CD production. Therefore, the enhancement

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the yields of CGTases produced by recombinant E. coli and recombinant B. subtilis were 20 and 300 times higher, respectively, than those of wild-type strains. In addition, Paloheimo et al. (1992) produced a profitable amount of CGTase using the recombinant B. subtilis strain, in which the yield reached 1.2 g/L—33 times the expression level of the parent B. circulans strain. Li et al. (2010b) expressed the gene encoding α-CGTase from P. macerans strain in E. coli as a C-terminal His-tagged protein. The activity of recombinant CGTase in the culture medium reached 22.5 U/mL after 90 h of induction, and it was approximately 42-fold higher than that from the parent strain.

Fig. 1. The 3-D structure of CGTase (PDB ID: 1CXK; Uitdehaag et al., 1999b). Domain A: Blue area; Domain B: red area; Domain C: green area; Domain D: yellow area; Domain E: gray area.

of CGTase production has attracted increasing interest. Successful CGTase production from native organisms via fermentation optimization has been reported. For example, Gawande and Patkar (1999) optimized the production of CGTase from Klebsiella pneumoniae pneumoniae AS-22 in flasks using a statistical experimental design approach and increased enzyme activity 9-fold compared to that in the basal medium. Rosso et al. (2002) combined conventional sequential techniques and statistical experimental design to optimize batch culture conditions for CGTase production with B. circulans DF 9R. The result indicated that the optimal medium not only increased CGTase activity but also avoided complicated nitrogen sources. However, heterologous expression of the CGTase gene in a suitable host is still regarded as a key factor in increasing CGTase production. A previous review indicates that approximately 50% of commercially produced CGTases are obtained through heterologous production (Qi and Zimmermann, 2005). As shown in Table 3, Wang et al. (2006) cloned cgt into the plasmid pYD1 and transformed it into Saccharomyces cerevisiae for expression. The expressed CGTase gave the yeast the capability to use starch directly as a sole carbon source and produce anticipated products such as CDs, glucose, and maltose. Paloheimo et al. (1992) successfully expressed cgt from B. circulans var. alkalophilus ATCC21783 in Bacillus subtilis. The amount of recombinant CGTase secreted in the culture medium was 14 times that of the parental strain in shake flask cultures. When cgt from P. macerans strain JFB05-01 was optimized in terms of codon usage and expressed in Bacillus megaterium, the CGTase activity reached 48.9 U/mL (Zhou et al., 2012). Among many alternative expression hosts, the bacterium E. coli is regarded as the most efficient and attractive for CGTase heterologous expression owing to its simplicity, low cost, known genetic properties, capacity to accommodate many foreign proteins, rapid culture, and high biomass and high protein yields (Assenberg et al., 2013), although it may not be the preferred production host (see Table 3).

3.1. Heterologous expression for overproduction Many examples have shown that the expression and production of CGTases can be enhanced by heterologous expression of the CGTase gene in a suitable host. As early as 1989, Schmid (1989) stated that

3.1.1. Molecular modification to improve CGTase secretion Simple heterologous expression usually cannot meet the requirement for overproduction of industrial enzymes. Therefore, combining molecular modifications with heterologous expression is necessary and efficient. Heterologous expression of recombinant proteins in E. coli is commonly problematic owing to the intracellular localization and formation of inactive inclusion bodies. Many attempts have been made to overcome this problem. Ong et al. (2008) expressed the CGTase gene from Bacillus sp. G1 in E. coli with the Bacillus sp. G1 signal peptide. They found that the excretory capability was accomplished by the Bacillus sp. G1 signal peptide and that the majority of active CGTase was secreted into the medium. Ayadi et al. (2011, 2012) constructed a recombinant enzyme by connecting CGTase from Paenibacillus pabuli US132 with the amylase signal peptide of Bacillus stearothermophilus and secretive lipase signal peptide of B. subtilis. When the recombinant enzymes above were expressed in E. coli, both improved the efficient secretion of CGTase into the culture medium as an active form, contrasting with the periplasmic production of native CGTase. Ismail et al. (2011) found that the mutant L -asparaginase II signal peptide could reduce the formation of intracellular inclusion bodies and improve the secretion of recombinant CGTase into the periplasmic space of E. coli. In addition, to overcome the problem of in vitro inclusion bodies, Hellman et al. (1995) investigated the recovery of active CGTase from inclusion bodies through expression of a signal sequence deletion mutant in E. coil, and the in vitro renaturation yield of active enzyme was up to 81% of the maximum activity from urea solubilization. Transport system modification is also an efficient approach to improving the secretion of recombinant CGTase. The hemolysin transport system mediates the release of CGTase into the extracellular medium when it is fused to the C-terminal 61 amino acids of HlyA. Low et al. (2010) engineered the hemolysin components via directed evolution (epPCR) to produce an improved secretion variant. The best variant showed a 2.1-fold increase in the secretion level compared to that in the wild-type strain. In addition, coexpression of folding accessory proteins, known as molecular chaperones and foldases, is an effective method for decreasing inclusion bodies in recombinant E. coli. Kim et al. (2005) expressed the B. macerans CGTase in E. coli with the molecular chaperones human peptidyl-proly1 cistrans isomerase, and reported that the amount of CGTase activity reached 1200 U/mL in fed-batch fermentation. 3.1.2. Molecular modification to improve CGTase overexpression Modifications of transcription and translation systems have also been investigated to overexpress recombinant CGTases. For instance, 3 DNA fragments starting from the nucleotide positions + 1, − 18, and −48 of the translation initiation site of the Bacillus sp. 1011 β-CGTase gene have been prepared and fused with 3 E. coli promoters (tac, trp, and PL). The combination of trp promoter and β-CGTase gene starting from nucleotide position − 48 in the presence of the inducer 3indolylacetic acid produces the maximum yield of β-CGTase, which is 5.5 times that of E. coli harboring the original plasmid and approximately 3 times higher than the extracellular production of parental Bacillus sp. 1011 (Kimura et al., 1990). Additionally, insertion of the regulatory sequence involved in the promotion of RNA transcription yields heterologous expression of CGTase that is 3.2-fold higher than that of the wild-

Table 2 The applications of CGTases from different sources. Product

Catalyst

Source*

Improved properties

Citation

Cyclodextrin (CD) production

α-CD

α-CGTase

Bacillus circulans

The specificity of α-CGTase

van der Veen et al. (2000c) Fujiwara et al. (1992a) Bender (1990) Hashimoto et al. (2001) Li et al. (2010b) Bender (1990) Kimura et al. (1987) Joergensen and Fulgsang, (2003) Itkor et al. (1990) Takada et al. (2003) Shim et al. (2004, 2007) Jemli et al. (2007) Jung et al. (2007) Shimoda et al. (2009)

β-CD

β-CGTase

Baking

γ-CD Bread-baking

Transglycosylation for improving carbohydrate properties

Glycosylated stevioside Glycosylation tocopherol

γ-CGTase CGTase β-CGTase CGTase Klebsiella pneumoniae and CGTase Xanthomonas campestris and CGTase Strophanthus gratus cell culture and CGTase Toruzyme 3.0 L (CGTase)

Hesperetin glycosides Curcumin βmaltooligosaccharides Glucosylation steroidal saponins Alkyl glucopyranosides

Bacillus mancerans Klebsiella pneumoniae Thermococcus sp. Paenibacillus macerans Bacillus circulans Alkalophilic Bacillus sp. Bacillus agaradhaerens Bacillus sp. Bacillus clarkii Alkalophilic Bacillus sp. Paenibacillus pabuli Alkalophilic Bacillus sp. –

The specificity of β-CGTase

The specificity of γ-CGTase Shelf life Improve loaf volume and decrease firmness of bread Alleviating bitters Water solubility and stability

–

Shimoda and Hamada (2010) Shimoda et al. (2007)

– –

Decreasing cytotoxicity

Wang et al. (2010)

CGTase

Bacillus macerans

Decreasing toxicity and biodegradable

Glycosyl-rutin

CGTase

Bacillus macerans

Solubility

Glycosyl-L-ascorb acid

CGTase

Inoxidizability and stability

Glucosyl-anhydro-Dfructose Arbutin-α-glycosides Glycosylated genistin Glycosyl glycerol

CGTase

Paenibacillus sp. Paenibacillus macerans Bacillus stearothermophilus

Solubility, stability and resitance to proteases

Svensson and Adlercreutz (2011) Go et al. (2007); Suzuki and Suzuki (1991) Jun et al. (2001) Zhang et al. (2011) Yoshinaga et al. (2003)

CGTase CGTase

Bacillus macerance Bacillus sp. Geobacillus stearothermophilus and Thermoanaerobacter sp. Bacillus ohbensis Bacillus macerans

Inhibitory effect on human tyrosinase Water solubility High-thermal stability, low heat-colorability, low Maillard reactivity, low hygroscopicity, Water-holding capacity, non-cariogenicity and low digestibility by rat intestinal enzymes Physiological value of assimilability High purity

Sato et al. (1992) Vetter et al. (1992)

CGTase CGTase CGTase

Bacillus circulans Bacillus sp. Bacillus stearothermophilus

Solubility Solubility Reactivities in a lipase-catalyzed esterification

Radu et al. (2006) Go et al. (2007) Nakano et al. (1992)

CGTase CGTase CGTase and glucoamylase CGTase

Bacillus macerans Bacillus macerans Bacillus stearothermophilus Thermoanaerobacter sp.

Specific glycosides of maltodextrins Reducing irritability and toxicity Reducing bitterness Stability

Yoon and Robyt (2006) Yoon et al. (2004) Kim et al. (2001) Martın et al. (2004)

CGTase

–

Anti-tumoral and insecticidal activity

Okada et al. (2007)

Glucosyl-inositol Linear and branched glucooligosaccharides Glycosyl-luteolin Glycosyl-bioflavonoids Glucosyltrimethylolpropane Glucosyltrimethylolethane Glucosyl-pentaerythritol Maltodextrin glycosides α-Salicin α-glucosylginsenosides Maltooligosyl fructofuranosides Glycosylated sucrose laurate

CGTase CGTase CGTase

Sugimoto et al. (2003) Li et al. (2005) Nakano et al. (2003)


Application

“*”: The source of cyclodextrin glucosyltransferase; “–”: unknown. 419

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Table 3 Heterologous expression of CGTases from different sources. WT strain

Recombinant strain

Optimizing strategy

CGTase expression and improvement

Citation

Bacillus macerans

Bacillus subtilis

Heterogeneous expression

CGTase gene clone and heterogeneous expression.

Klebsiella pneumoniae Bacillus stearothermophilus Alkalophilic Bacillus sp. Bacillus subtilis

Escherichia coli



Escherichia coli



Takano et al. (1986) Binder et al. (1986) Sugimoto et al. (1986)

Escherichia coli



Coexpression of a molecular chaperone (human PPIase) Fusion with a α-agglutinin gene

Active CGTase activity reached 1200 U/mL.

Thermotolerant Escherichia coli Paenibacillus sp. Pyrococcus furiosus Escherichia coli


Paenibacillus pabuli Bacillus sp.

Escherichia coli

Culture parameters optimization

Escherichia coli

Heterogeneous expression and using Bacillus sp. G1 signal peptide Supplementation of glycine and calcium

Charoensakdi et al. (2007) Lee et al. (2007) Recombinant CGTase reached 35 U/mL. Jemli et al. (2008) Increase the CGTase secreted into the medium. Ong et al. (2008) Enhances the extracellular secretion of α-CGTase in E. coli. Li et al. (2009a) The fusion enzyme has Increased the expression level and 2.7 fold higher Kaulpiboon maximum specific activity than the non-fusion form. et al. (2010b)

Escherichia coli

Bacillus sp.

Escherichia coli

Bacillus circulans

Escherichia coli

Bacillus circulans

Escherichia coli

Bacillus circulans

Bacillus subtilis

Bacillus macerans

Bacillus subtilis

Bacillus macerans

Escherichia coli

Brevibacillus brevis Escherichia coli Bacillus macerans

Bacillus macerans Bacillus circulans

Paenibacillus macerans Paenibacillus sp.

Paenibacillus macerans Paenibacillus macerans Paenibacillus macerans Bacillus sp.

Escherichia coli and Bacillus subtilis Escherichia coli Saccharomyces cerevisiae

Escherichia coli Escherichia coli

Kaneko et al. (1988) Heterogeneous expression CGTase gene clone and heterogeneous expression. Kato and Horikoshi (1986) Fusion with tac, trp and PL promoters CGTase yield increased to 5.5 times than the original E. coli plasmid and Kimura et al. 3.3 times higher than the parental Bacillus sp. (1990) Fusion CGTase gene with alkaline phosphatase and Enable the absorption of the fusion proteins directly from the culture Hellman and α-amylase medium onto α-CD coupled agarose. Mantsala (1992) Signal sequence deletion phosphatase and αRecovery of CGTase activity from inclusion bodies. Hellman et al. amylase (1995) Heterogeneous expression Extracellular expression 1.2 g/L (33 times of the WT). Paloheimo et al. (1992) Heterogeneous expression CGTase gene clone and heterogeneous expression. Lin and Jeang (1998) Heterogeneous expression of WT and mutants Only WT and E344D CGTases became soluble when heterogeneous Jeang et al. E344D, E344K, E344L. expressed. (1999) Control the temperature and mannitol Increase 34-fold in biologically active soluble form. Kim et al. concentration (1999) Comparation of the characterization of CGTases Obtained the differences of the characterization of CGTases expressed by Jeang et al. expressed by different host strains different host strains. (2005)

Codon optimization

Escherichia coli

Fusion with thioredoxin, hexa-histidine and Sprotein and a proline-rich peptide at the Cterminus of CGTase Heterogeneous expression

Escherichia coli

Supplementation of glycine

Escherichia coli

Supplementation of medium additives

Escherichia coli

Using hemolysin transport system and further creating Hly variants by error-prone PCR With a mutant L-asparaginase II signal peptide

Paenibacillus pabuli Bcaillus sp.

Escherichia coli

Paenibacillus macerans Paenibacillus macerans Paenibacillus macerans Paenibacillus illinoisensis Paenibacillus pabuli Paenibacillus pabuli Bcaillus sp.

Escherichia coli

Escherichia coli

New plasmid designing and optimization of culture conditions A fed-batch fermentation strategy

Bacillus megaterium Escherichia coli

Systematic codon optimization

Escherichia coli


Escherichia coli

Fusion with Bacillus stearothermophilus amylase signal peptide Fusion with Bacillus subtilis lipase signal peptide

Escherichia coli Escherichia coli

Systematic codon optimization

Insertion of regulatory sequences involved in the promotion of RNA transcription

Obtained surface-anchored CGTase which enable the yeast to utilize starch as a sole carbin source and produce cyclodextrins, as well as glucose and maltose. Shorten the fermentation time, increase CGTase thermostability compared with WT. Overexpression of CGTase.

Overexpression and increasing 42-fold activity than the parent strain.

Kim et al. (2005) Wang et al. (2006)

Li et al. (2010b) Enhances the extracellular secretion of α-CGTase in E. coli. Li et al. (2010a) Enhances the extracellular secretion of α-CGTase in E. coli. Ding et al. (2010) The recombinant protein was secreted in large quantities extracellulary. Low et al. (2010) Improve the secretion of recombinant CGTase and the viability of E. coli. Ismail et al. (2011) Extracellular CGTase activity reached 69.15 U/mL, resulting in a 3.45-fold Low et al. increase compared to the initial conditions. (2011) Extracellular CGTase activity reached 275.3 U/mL. Cheng et al. (2011) Recombinant CGTase activity reached 48.9 U/mL. Zhou et al. (2012) CGTase yield reached 2520 mg/L and extracelluar activity reached Liu et al. 55.3 U/mL. (2012) Purification and properties of recombinant β-CGTase. Lee et al. (2013) Improve the extracellular secretion of CGTase efficiently. Ayadi et al. (2011) Ayadi et al. (2012) Recombinant CGTase activity increased to 3.2-fold compared to the WT. Ramli et al. (2013)


type producer (Bacillus sp. NR5UPM) (Ramli et al., 2013). Systematic codon usage optimization is often used to improve efficient translation in some bacterium. In our previous work, the α-CGTase gene from P. macerans was optimized according to the codon preference of the expression host to achieve overexpression. When the codon-optimized α-CGTase gene was expressed in E. coli BL 21(DE3), extracellular α-CGTase activity was 326% that of wild-type CGTase (Liu et al., 2012), and when it was expressed in B. megaterium, α-CGTase activity reached 48.9 U/mL, which was nearly 98-fold greater than that of the parent strain (Zhou et al., 2012). In addition, Kaulpiboon et al. (2010b) constructed a recombinant CGTase gene by fusing thioredoxin, hexa-histidine, and S-protein at the N-terminus and a proline-rich peptide at the C-terminus, increasing expression 2.7-fold that of the non-fusion form. 3.1.3. Cultivation strategy optimization to improve overproduction of CGTase Optimization of cultivation strategies is another efficient method for CGTase overproduction. Supplementation of cultures with medium additives such as glycine and calcium increases the extracellular activity of α-CGTase (Ding et al., 2010; Li et al., 2009a, 2010a). Cheng et al. (2011) increased the extracellular activity of α-CGTase to 275.3 U/mL with high-cell-density cultivation of E. coli in fed-batch fermentation. In addition, Low et al. (2011) optimized culture conditions through central composite design and increased extracellular CGTase activity to 3.45fold that under the initial conditions. Jemli et al. (2008) also increased CGTase production by using 2 × TY medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl) instead of LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) and shifting the fermentation temperature from 37 °C to 19 °C. 3.2. Heterologous expression to improve properties Certain CGTase properties have been improved after heterologous expression. For example, the B. macerans CGTase gene has been expressed in E. coli (Jeang et al., 1999) and B. subtilis (Jeang et al., 2005; Lin and Jeang, 1998), demonstrating that different CGTase activities and properties occur depending on the host. β-CD coupling activity of the CGTase expressed in E. coli is reportedly 14-fold higher than that of enzymes from other strains, whereas α-CD coupling activities were the same among these CGTases (Jeang et al., 2005). Charoensakdi et al. (2007) isolated the thermotolerant Paenibacillus sp. T16 containing a CGTase gene from hot springs in northern Thailand and further expressed the CGTase gene in E. coli (JM109). This expression revealed significant differences in pH, thermal stability, and kinetic parameters, especially in the presence of high starch concentration, in which the thermal stability of recombinant CGTase was higher than that of the wild-type. The recombinant enzyme was also more stable at higher temperature and lower pH, with a Km lower than that of wild-type CGTase. Wang et al. (2006) successfully expressed CGTase with α-agglutinin at the N-terminus on the extracellular surface of S. cerevisiae and obtained an immobilized enzyme, surface-anchored CGTase, which gave the yeast the capability to use starch directly as a sole carbon source and produce the anticipated product, CD. Interestingly, Duan et al. (2013) enhanced the CD production by synchronously utilizing of recombinant isoamylase and α-CGTase. Under the optimum conditions (10 U α-CGTase and 48 U isoamylase per gram of substrate with reaction temperature of 30 °C and pH 5.6), the total CD titer converted from potato starch reached 84.6% (w/w), which was 31.2% higher than the transformation with α-CGTase. 4. Molecular engineering of CGTase to improve properties Improved adaptation of CGTases for various applications in the synthesis of CDs and other compounds requires molecular modification of

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native CGTases. Herein, we review some common and novel protein engineering strategies for the molecular modification of CGTases (Fig. 2). 4.1. Site-directed mutagenesis Site-directed mutagenesis is an efficient method for engineering essentially any property of enzymes based on their crystal structures or 3D simulation structures. Recently, site-directed mutagenesis has been applied extensively to improve CGTase properties. 4.1.1. Site-directed mutagenesis for improving the product specificity of CGTases The main application of CGTases is the production of CDs through the cyclization reaction. These enzymes primarily produce 3 types of CDs, which differ in their degree of polymerization: CD6, CD7, and CD8. Commonly, the products catalyzed by CGTases are mixture of these CDs, and their quantities and size specificities vary greatly among CGTases from different organisms. This variation increases the difficulty in CD separation and purification. Therefore, the improvement of the product specificity of a CGTase using a molecular modification approach is increasingly important. Table 4 shows that many sites have been targets of site-directed mutagenesis for investigations of CD specificity. Some mutants have contributed to improving CD6 specificity: G180L, Y167F, and N193L (Leemhuis et al., 2002), belonging to the − 6 subsite; Y89D (van der Veen et al., 2000c) and N94S (Kim et al., 1997), belonging to the − 3 subsite; S146P (van der Veen et al., 2000c), belonging to the − 7 subsite; and D197H and F284K (Wind et al., 1998). Some variants have been regarded as preferential in the formation of β-CD: Y89G (van der Veen et al., 2000c) and Y89F (Kim et al., 1997) (−3 subsite), N327D (Wind et al., 1998) (− 1 subsite), Y100F (Kim et al., 1997), and E264Q (Nakagawa et al., 2006). Furthermore, certain mutants enhance γ-cyclization activity: W188Y (Sin et al., 1994), Y195W and L194T (+1 subsite) (Parsiegla et al., 1998), Y195L (Nakamura et al., 1994), H43T (Goh et al., 2007) (−3 subsite of β-CGTase G1), H43T/Y87F (Goh et al., 2009), and A223K/R/H (Nakagawa et al., 2006). In addition, Y267W (Yamamoto et al., 2000), R47Q (van der Veen et al., 2000b), and D371R (Wind et al., 1998) (−3 subsite) improve both β- and γ-cyclization activities. These results show that the − 6 and − 3 substrate binding subsites play important roles in the cyclization reaction. 4.1.2. Site-directed mutagenesis for improving the substrate specificity of CGTases Recently, we have become interested in using a cheap and soluble substrate to replace expensive α-CD and insoluble β-CD as the glycosyl donor for AA-2G synthesis. The substrate specificity of CGTase toward maltodextrin, an ideal succedaneum, was enhanced by a series of sitedirected saturation mutagenesis. First, the saturation mutagenesis of lysine 47 (belonging to the −3 subsite) showed that the mutants K47L, K47F, K47V, and K47W improve maltodextrin specificity for AA-2G production. With K47L in particular, AA-2G yield increased 64.2% compared to that of the wild-type CGTase (Han et al., 2013c). Then other amino acid residues of the −3 subsite (tyrosine 89, asparagine 94, and aspartic acid 196) were targeted for saturation mutagenesis. The result showed that the combination mutant K47L/Y89F/ N94P/D196Y produced the highest AA-2G titer of 2.23 g/L with maltodextrin as the glycosyl donor. This titer was an increase of 85.8% compared with that of the wild-type CGTase (Liu et al., 2013). The + 2 subsite amino acid residues (tyrosine 195, tyrosine 260, and glutamine 265) were also engineered via saturation mutagenesis to improve maltodextrin specificity, with the mutant Y260R/Q265K/Y195S producing an AA-2G yield of 1.92 g/L—60% higher than that of the wild-type CGTase (1.20 g/L) (Han et al., 2013b). These results indicated that the residues at the −3 and +2 subsites are related to the differences in substrate specificities.

422


Fig. 2. The approaches of molecular modification of CGTases to improve properties.

4.1.3. Site-directed mutagenesis for improving the hydrolysis activity of CGTases The hydrolysis activity of CGTase is often much lower than the transglycosylation activity despite its belonging to the α-amylase family (van der Veen et al., 2000d). Improving hydrolysis activity to transfer CGTase to a hydrolytic enzyme has attracted strong attention in recent years. Van der Veen et al. (2001) found that the hydrophobicity of phenylalanine 183 and phenylalanine 259 limits the hydrolyzing activity of the CGTase. Hydrolysis could be enhanced by making the residues more polar—for example, F183S, F183N, F259N, and F259S—which concomitantly resulted in lower transglycosylation activity. In particular, the double mutant F183S/F259N was constructed and the resulting CGTase preferred hydrolysis over cyclization (15:1). Nakamura et al. (1994) have also indicated that Phe-183 and Phe-259 of the CGTase play critical roles in cyclization through cooperative involvement in acceptor binding. Similarly, Fujiwara et al. (1992b) have suggested the hydrophobic aromatic amino acid residues at position 255 of CGTase from B. stearothermophilus are important not only for cyclization but also for the hydrolysis reaction. In addition, Kelly et al. (2007) constructed a triple mutant CGTase (A231V/F260W/F184Q) via saturation mutagenesis, which converted the enzyme into an α-amylase-like hydrolase with high hydrolytic activity and no CD production capacity.

4.1.4. Site-directed mutagenesis for improving the thermostability of CGTases Leemhuis et al. (2004) have suggested that the mutant CGTase N188D/K192R strongly enhances the thermostability (half-life at 60 °C) of CGTase by introducing a salt bridge. Kelly et al. (2009) have found that CGTases from different sources and with different structures generally have different thermostabilities, and ionic interaction in CGTase contributes to the thermostability of this kind of enzyme. Goh et al. (2012a) demonstrated that the calcium-binding regions of CGTase of Bacillus sp. G1 contribute to thermostability. Four CGTase variants (S182G, S182E, N132R, and N28R) were constructed, and the residues at the 182 position (S182G and S182E) located adjacent to the Ca-I site and the active site cleft, displayed enhanced thermostability. The Asn28 residue at calcium-binding site II significantly affected CGTase specific activities but not thermostability. The N132R mutant completely disrupted CGTase activity. The thermostability of CGTase is also improved by B domain replacement. For example, Goh et al. (2012b) replaced the B domain of Bacillus sp. G1 CGTase H43T/Y87F with the similar domain of a thermostable CGTase from B. stearothermophilus ET1. As a result, the half-life of the mutant at 60 °C was approximately 2.5-fold that of the parent CGTase. Moreover, thermostability can also be improved through computer simulation prediction using a pseudoamino acid composition model (PseAAC) (Wang et al., 2011).

Table 4 Molecular modification strategies of CGTases to improve the properties in applications. Modification strategy

Improving property

Positions

Improvements

Site-directed mutagenesis

Product specificity

Y100W, W191Y, Y267W, W191F, Y267F

Mutant Y267W improved the β- and γ-cyclization activities.

D197H, F284K, N372D, D371R Y167F, G179L, G180L, N193G, N193L, G179L/G180L R47L, R47Q Y89G, Y89D, S146P, Y89D/S146P Saturation mutagenesis of W188 H43T H43T/Y87F D229A, Y195W, L194T

D229N, E257Q, E264Q, D328N Q179L and Q179G

Substrate specificity

A223D, A223E, A223S, A223K, A223R, A223H, G255D, G255E, G255S, G255K, G255R and G255H Saturation mutagenesis of K47 Combination saturation mutagenesis of Y195, Y260 and Q265 Combination saturation mutagenesis of K47, Y89, N94 and D196

Hydrolysis ability

E263A, K232L, K232Q, F259N, F183S, F183N, F259S, F183S/F259N

Thermostability

A231V/F260W/F184Q S182G, S182E, N192R and N28R N188D/K192R

Cyclization

B domain replacement with the thermostable CGTase S77P and W239R

Hydrolysis ability

A230V

Product specificity

Deletion of loop 145–151 (△(145–151)D)

Catalytic function

Deletion of loop 139–144 (△(139–144)D)

Domain fusing

Catalytic function Substrate specificity

Molecular imprinting

Dextrinizing and cyclization acitivities Product specificity

Deletion of loop 154–160 (△(154–160)D) Construction of two chimeras (CGT△E-CBMAmy and CGT-CBMAmy) by fusing the CGTase and CBM domain of amylase Construction various chimeras of different combinations of domains and part of domains A/B between the α-CGTase and β-CGTase Molecular imprinting with cyclomaltododecaose

Random mutations (error-prone PCR)

Fragment deletion

Yamamoto et al. (2000) D197H and F284K improved the α-cyclization activity, D371R improved the β- and γ-cyclization Wind et al. (1998) activities, N327D improved the β- cyclization activity. G180L, Y167F and N193L improved the α-cyclization activity, G179L decreased the α-cyclization Leemhuis et al. activity, N193G and G179L/G180L decreased the β-cyclization activity. (2002) R47Q improved β- and γ-cyclization activities. van der Veen et al. (2000a,b) Y89G improved the β-cyclization activity, Y89D, S146P, Y89D/S146P improved the α-cyclization van der Veen et al. activity. (2000c) W188Y improved the γ-cyclization activity. Sin et al. (1994) Improved the γ-CD product speicificity. Goh et al. (2007) Improved the γ-CD product speicificity. Goh et al. (2009) Y195W, L194T improved the γ-cyclization activity. Parsiegla et al. (1998) N94S improved the α-cyclization activity, Y89F and Y100F improved the β-cyclization activity. Kim et al. (1997) Y195L improved the γ-cyclization activity while lost the α-cyclization activity and others lost the Nakamura et al. cyclization activity. (1994) E264Q improved the β-cyclization activity and others lost the cyclization activity. Nakagawa et al. (2006) Q179L decreased the α-cyclization and β-cyclization activity while Q179G remained unchanged Costa et al. (2012) on the product ratio. A223K, A223R, A223H enhanced the γ-cyclization activity while other mutants decreased the γ- Nakagawa et al. cyclization activity. (2006) K47L, K47F, K47V and K47W improved the maltodextrin specificity for AA-2G production. Han et al. (2013c) Y195S, Y260R and Q265K and their combination mutants improved the maltodextrin specificity Han et al. (2013b) for AA-2G production. K47L, Y89F, N94P and D196Y and their combination mutants improved the maltodextrin Liu et al. (2013) specificity for AA-2G production. F259N, F183S, F183N, F259S and F183S/F259N improved the hydrolysis activity. van der Veen et al. (2001) A231V/F260W/F184Q improved the hydrolysis activity. Kelly et al. (2007) S182G and S182E enhanced the thermostability. Goh et al. (2012a) Strongly enhanced the thermostability. Leemhuis et al. (2004) Improved the half-time at 60 °C to about 2.5-fold of WT. Goh et al. (2012b) Elimination of the competing hydrolysis and coupling side reactions to improve the cyclization Kelly et al. (2008) reaction. A230V increased the hydrolytic activity 90-fold. Leemhuis et al. (2003b) △(145–151)D improved the γ-cyclization activity Parsiegla et al. (1998) The loop 139–144 of CGTase G1 plays important role on cyclization and coupling reaction, while Goh et al. (2009) has less effect on hydrolysis. This mutant improved cyclization and CD-forming conversion yield. Lee et al. (2003) Two chimeras CGT△E-CBMAmy and CGT-CBMAmy improved the soluble starch specificity for AA- Han et al. (2013a) 2G production. Different chimeras showed different dextrinizing and cyclization activities. Rimphanitchayakit et al. (2005) Kaulpiboon et al. Increased the proportion of CD12 and large CD.s (2010a)


N94S, Y89F, Y100F Y195L, F183L, F259L, F183L/F259L, Y283L

Citation

423

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4.2. Random mutation (error-prone polymerase chain reaction, epPCR) epPCR is one of the most frequently used directed evolution methods, and it has been used for molecular modification of CGTases. Kelly et al. (2008) applied random mutations throughout the CGTase gene via epPCR to reduce the competing hydrolysis reaction while maintaining cyclization activity. As a result, 2 mutants, S77P and W239R, were found to lower hydrolytic activity up to 15-fold while retaining cyclization activity. Leemhuis et al. (2003b) carried out epPCR mutagenesis and screened B. circulans strain 251 CGTase mutants with increased hydrolytic activity. The results showed that the hydrolytic activity of the A230V mutant increased to 90-fold that of wildtype CGTase, and the mutant converted the CGTase into a starch hydrolase. Shim et al. (2004) performed epPCR on CGTase from alkalophilic Bacillus sp. to improve its properties as an antistaling enzyme. The random mutant CGTase[3–18] slowed the retrogradation of the starch fraction in baked goods, which can lengthen the shelf life of foods. 4.3. Fragment deletion mutagenesis Parsiegla et al. (1998) constructed a variant that deleted loop 145–151 of CGTase from B. circulans strain no. 8 with a replacement of aspartic acid

151. The deletion mutant Δ(145–151)D increased γ-cyclization activity and improved CD8 product specificity. The crystal structure of the variant suggested that the deletion can produce more space for the bound glucosyl chain and the aspartate could provide a shortcut to help bind saccharides. Goh et al. (2009) expected to improve the product specificity of β-CGTase G1 by constructing mutant Δ(139–144) in which loop 139–144 was deleted. However, the result showed this mutant significantly decreased the cyclization and coupling activity while increasing hydrolysis activity. This outcome revealed that loop 139–144 (within the –7 subsite) plays a key role in cyclization and coupling reactions and has a smaller effect on hydrolysis. However, another deletion mutant, Δ(154–160), within the –7 subsite of β-CGTase from Bacillus firmus var. alkalophilus increased cyclization and improved the conversion into CDs from 28.6% to 39% (Lee et al., 2003). 4.4. Domain chimera To improve the soluble starch substrate specificity for AA-2G production, Han et al. (2013a) constructed 2 chimeric enzymes by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBMAmy) to CGTase from P. macerans. One had CBMAmy at the C-terminal region of CGTase, and the other was obtained by

Fig. 3. The schematic presentation of the perspective of CGTase researches and applications.


replacing the E domain of CGTase with CBMAmy. Both chimeras showed improved transformation efficiency of soluble starch, which could be a cheap and easily soluble glycosyl donor for the synthesis of AA-2G. The enhancement of soluble starch specificity may be related to changes in substrate binding capability and the substrate binding sites between the CBM and the starch granule via structure modeling of CGTase and molecular docking with malt disaccharide (Han et al., 2013a). In addition, Rimphanitchayakit et al. (2005) constructed various chimeras with combinations of domains and parts of domains A and B between the α-CGTase from B. macerans IAM1243 and β-CGTase from B. circulans A11 to analyze dextrinization and CD formation. They found that different chimeras showed different dextrinization and cyclization activities, indicating the possible function of various domain regions. 4.5. Molecular imprinting CGTases often catalyze the formation of a mixture of CDs from starch via an intramolecular transglycosylation reaction. To manipulate the production specificity of CD8, Kaulpiboon et al. (2007) applied molecular imprinting and immobilization methodologies to prepare the CGTases of Paenibacillus sp. A11 and B. macerans. As a result, the efficiency of the cross-linked imprinted enzymes for CD8 synthesis was increased to 10-fold that of the native enzymes. Subsequently, they preferentially synthesized cyclomaltododecaose via molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose as the template molecule, which proved to be an effective means for increasing the yield of large-ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. 5. Conclusions and perspective CGTase is generally regarded as an important biocatalyst with multiple functions related to its 4 catalytic reactions, which have wide applications in industrial engineering and life sciences. Therefore, the future of CGTases will attract more attention. Here, a simple perspective of CGTases (as shown in Fig. 3) may provide some future directions of CGTases. The many investigations of CGTases have provided a better understanding of the crystal structure, reaction mechanisms, engineering, and industrial applications of this key enzyme. In recent years, an increasing number of new microorganisms have been found to produce novel CGTases with novel properties such as high temperature and high pH stabilities that could be applied under extreme conditions. These discoveries provide a clear direction for screening microorganisms in wild and for enhancing CGTase performance by using properties such as substrate conversion, product specificity, stability, and specific activities. However, some novel CGTases may have special properties (e.g., strong stability in extreme temperature or pH environments) and functions (e.g., high product or substrate specificities) that remain to be discovered and investigated. Therefore, genome and metagenome sequencing may be used to perform bioinformatics analysis and data mining of unexplored CGTases (or CGTase-like proteins) in the future. We still expect that CGTases will find more widespread applications. For example, Wang et al. (2006) have provided the new insight that CGTase can change the conditions of microbial growth (using simple and cheap starch to replace expensive cultures as carbon sources). Moreover, another prospect is the investigation of CGTase transglycosylation to improve carbohydrate properties with inexpensive substrate—for example, AA-2G synthesis with cheap glycosyl donors (Han et al., 2012). Although many marvelous successes in CGTase overproduction have occurred during the past 2 decades, extensive work remains to be done to improve CGTase production. Many literature reports focus mainly on the secretion of recombinant CGTases, whereas few describe the transcription and translation of the CGTase gene. Therefore, systematic upstream and downstream modification and optimization are necessary for the

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heterologous expression of CGTase. For instance, transcription factors can be modified to improve the transcriptional level, the transcription level can be enhanced through codon optimization, and the secretion level can be improved through signal peptide optimization. Finally, various molecular modification strategies have been undertaken to improve the properties of CGTase, thereby reducing production costs and improving catalysis efficiency. However, these achievements remain limited and are far from the ultimate requirements desired. Therefore, considerable effort and work remain to improve CGTase production and properties. Acknowledgments This work was supported financially by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (111-2-06) Fundamental Research Funds for the Central Universities (JUSRP211A29), 863 Program (2011AA100905), and 973 Program (2012CB720806). References Abelian V, Afyan K, Avakian Z, Melkumyan A, Afrikian E. Cyclomaltodextrin glucotransferases from thermophilic actinomycetes. Biokhimiya 1995;60:1223–30. Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 2013;23:393–402. Atanasova N, Petrova P, Ivanova V, Yankov D, Vassileva A, Tonkova A. Isolation of novel alkaliphilic bacillus strains for cyclodextrin glucanotransferase production. Appl Biochem Biotechnol 2008;149:155–67. Ayadi DZ, Kammoun R, Jemli S, Bejar S. Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design. Biologia 2011;66:945–53. Ayadi DZ, Kammoun R, Jemli S, Chouayekh H, Bejar S. Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium. Indian J Exp Biol 2012;50:72–9. Bautista V, Esclapez J, Perez-Pomares F, Martinez-Espinosa RM, Camacho M, Bonete MJ. Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei. Extremophiles 2012;16:147–59. Bender H. Studies of the mechanism of the cyclisation reaction catalysed by the wildtype and a truncated α-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al, and the β-cyclodextrin glycosyltransferase from Bacillus circulans strain 8. Carbohydr Res 1990;206:257–67. Binder F, Huber O, Bock A. Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression. Gene 1986;47:269–77. Charoensakdi R, Murakami S, Aoki K, Rimphanitchayakit V, Limpaseni T. Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand. J Biochem Mol Biol 2007;40:333–40. Cheng J, Wu D, Chen S, Chen J, Wu J. High-level extracellular production of α-cyclodextrin glycosyltransferase with recombinant Escherichia coli BL21 (DE3). J Agric Food Chem 2011;59:3797–802. Costa H, Distefano AJ, Marino-Buslje C, Hidalgo A, Berenguer J, Bonino MBD, et al. The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase. Appl Microbiol Biotechnol 2012;94:123–30. Dalmia BK, Schutte K, Nikolov ZL. Domain E of Bacillus macerans cyclodextrin glucanotransferase: an independent starch-binding domain. Biotechnol Bioeng 1995;47:575–84. Dauter Z, Dauter M, Brzozowski AM, Christensen S, Borchert TV, Beier L, et al. X-ray structure of Novamyl, the five-domain “maltogenic” alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7 A resolution. Biochemistry 1999;38:8385–92. Ding R, Li Z, Chen S, Wu D, Wu J, Chen J. Enhanced secretion of recombinant α-cyclodextrin glucosyltransferase from E. coli by medium additives. Process Biochem 2010;45:880–6. Duan X, Chen S, Chen J, Wu J. Enhancing the cyclodextrin production by synchronous utilization of isoamylase and alpha-CGTase. Appl Microbiol Biotechnol 2013;97: 3467–74. Fujiwara S, Kakihara H, Woo K, Lejeune A, Kanemoto M, Sakaguchi K, et al. Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme. Appl Environ Microbiol 1992a;58:4016–25. Fujiwara S, Kakihara H, Sakaguchi K, Imanaka T. Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability. J Bacteriol 1992b;174:7478–81. Gawande B, Patkar A. Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS‐22. Biotechnol Bioeng 1999;64:168–73. Go YH, Kim TK, Lee KW, Lee YH. Functional characteristics of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. BL-31 highly specific for intermolecular transglycosylation of bioflavonoids. J Microbiol Biotechnol 2007;17:1550–3. Goh KM, Mahadi NM, Hassan O, Abdul Rahman RNZR, Illias RM. The effects of reaction conditions on the production of γ-cyclodextrin from tapioca starch by using a novel recombinant engineered CGTase. J Mol Catal B Enzym 2007;49:118–26.

426


Goh KM, Mahadi NM, Hassan O, Rahman RNZRA, Illias RM. A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity. J Mol Catal B Enzym 2009;57:270–7. Goh PH, Illias RM, Goh KM. Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability. Int J Mol Sci 2012a;13:5307–23. Goh PH, Illias RM, Goh KM. Domain replacement to elucidate the role of B domain in CGTase thermostability and activity. Process Biochem 2012b;47:2123–30. Haga K, Kanai R, Sakamoto O, Aoyagi M, Harata K, Yamane K. Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011. J Biochem 2003;134:881–91. Han R, Liu L, Li J, Du G, Chen J. Functions, applications and production of 2-O-Dglucopyranosyl-L-ascorbic acid. Appl Microbiol Biotechnol 2012;95:313–20. Han R, Li J, Shin HD, Chen RR, Du G, Liu L, et al. Carbohydrate-binding module-cyclodextrin glycosyltransferase fusion enables efficient synthesis of 2-o-d-glucopyranosyl-L-ascorbic acid with soluble starch as the glycosyl donor. Appl Environ Microbiol 2013a;79:3234–40. Han R, Liu L, Shin HD, Chen RR, Li J, Du G, et al. Systems engineering of tyrosine 195, tyrosine 260, and glutamine 265 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance maltodextrin specificity for 2-O-(D)-glucopyranosyl-(L)-ascorbic acid synthesis. Appl Environ Microbiol 2013b;79:672–7. Han R, Liu L, Shin HD, Chen RR, Du G, Chen J. Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-Dglucopyranosyl-L-ascorbic acid (AA-2G). Appl Microbiol Biotechnol 2013c;97: 5851–60. Harata K, Haga K, Nakamura A, Aoyagi M, Yamane K. X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 Å resolution. Acta Crystallogr D Biol Crystallogr 1996;52: 1136–45. Hashimoto Y, Yamamoto T, Fujiwara S, Takagi M, Imanaka T. Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001. J Bacteriol 2001;183:5050–7. Hellman J, Mantsala P. Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase. J Biotechnol 1992;23:19–34. Hellman J, Lassila P, Mantsala P. In vitro refolding of cyclomaltodextrin glucanotransferase from cytoplasmic inclusion bodies formed upon expression in Escherichia coli. Protein Expr Purif 1995;6:56–62. Ibrahim AS, Al-Salamah AA, El-Tayeb MA, El-Badawi YB, Antranikian G. A novel cyclodextrin glycosyltransferase from alkaliphilic Amphibacillus sp. NPST-10: purification and properties. Int J Mol Sci 2012;13:10505–22. Ishii N, Haga K, Yamane K, Harata K. Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 Å resolution. J Biochem 2000;127:383–91. Ismail NF, Hamdan S, Mahadi NM, Murad AM, Rabu A, Bakar FD, et al. A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli. Biotechnol Lett 2011;33: 999–1005. Itkor P, Tsukagoshi N, Udaka S. Nucleotide sequence of the raw-starch-digesting amylase gene from Bacillus sp. B1018 and its strong homology to the cyclodextrin glucanotransferase genes. Biochem Biophys Res Commun 1990;166:630–6. Janeček Š. Parallel β/α-barrels of α-amylase, cyclodextrin glycosyltransferase and oligo-1, 6-glucosidase versus the barrel of β-amylase: evolutionary distance is a reflection of unrelated sequences. FEBS Lett 1994;353:119–23. Jeang CL, Wung CH, Chang BY, Yeh SS, Lour DW. Characterization of the Bacillus macerans cyclodextrin glucanotransferase overexpressed in Escherichia coli. Proc Natl Sci Counc Repub China B 1999;23:62–8. Jeang CL, Lin DG, Hsieh SH. Characterization of cyclodextrin glycosyltransferase of the same gene expressed from Bacillus macerans, Bacillus subtilis, and Escherichia coli. J Agric Food Chem 2005;53:6301–4. Jemli S, Messaoud EB, Ayadi-Zouari D, Naili B, Khemakhem B, Bejar S. A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: purification, properties and potential use in bread-making. Biochem Eng J 2007;34:44–50. Jemli S, Ben Messaoud E, Ben Mabrouk S, Bejar S. The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme. J Biomed Biotechnol 2008;2008:692573. Joergensen PL, Fulgsang CC. CGTase and DNA sequence encoding same. EP Patent 1,303,633; 2003. Jun HK, Bae KM, Kim SK. Production of 2-O-alpha-D-glucopyranosyl L-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp. Biotechnol Lett 2001;23:1793–7. Jung S-W, Kim T-K, Lee K-W, Lee Y-H. Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic Bacillus sp. BL-12 and intermolecular transglycosylation of stevioside. Biotechnol Bioproc Eng 2007;12:207–12. Kanai R, Haga K, Yamane K, Harata K. Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin at 2.0 Å resolution. J Biochem 2001;129:593–8. Kanai R, Haga K, Akiba T, Yamane K, Harata K. Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: substrate binding and arrangement of the catalytic site. Protein Sci 2004;13:457–65. Kaneko T, Hamamoto T, Horikoshi K. Molecular cloning and nucleotide sequence of the cyclomaltodextrin glucanotransferase gene from the alkalophilic Bacillus sp. strain no. 38-2. J Gen Microbiol 1988;134:97–105. Kato T, Horikoshi K. Cloning and expression of the Bacillus subtilis No. 313 γ-cyclodextrin forming CGTase gene in Escherichia coli. Agric Biol Chem 1986;50:2161–2.

Kaulpiboon J, Pongsawasdi P, Zimmermann W. Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with γ‐cyclodextrin. FEBS J 2007;274:1001–10. Kaulpiboon J, Pongsawasdi P, Zimmermann W. Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose. J Mol Recognit 2010a;23:480–5. Kaulpiboon J, Prasong W, Rimphanitchayakit V, Murakami S, Aoki K, Pongsawasdi P. Expression and characterization of a fusion protein-containing cyclodextrin glycosyltransferase from Paenibacillus sp. A11. J Basic Microbiol 2010b;50:427–35. Kelly RM, Leemhuis H, Dijkhuizen L. Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies. Biochemistry 2007;46:11216–22. Kelly RM, Leemhuis H, Rozeboom HJ, van Oosterwijk N, Dijkstra BW, Dijkhuizen L. Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution. Biochem J 2008;413:517–25. Kelly RM, Dijkhuizen L, Leemhuis H. The evolution of cyclodextrin glucanotransferase produc specificity. Appl Microbiol Biotechnol 2009;84:119–33. Kim YH, Bae KH, Kim TJ, Park KH, Lee HS, Byun SM. Effect on product specificity of cyclodextrin glycosyltransferase by site-directed mutagenesis. Biochem Mol Biol Int 1997;41:227–34. Kim MH, Lee JK, Kim HK, Sohn CB, Oh TK. Overexpression of cyclodextrin glycosyltransferase gene from Brevibacillus brevis in Escherichia coli by control of temperature and mannitol concentration. Biotechnol Tech 1999;13:765–70. Kim Y-H, Lee Y-G, Choi K-J, Uchida K, Suzuki Y. Transglycosylation to ginseng saponins by cyclomaltodextrin glucanotransferases. Biosci Biotechnol Biochem 2001;65:875–83. Kim SG, Kweon DH, Lee DH, Park YC, Seo JH. Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli. Protein Expr Purif 2005;41:426–32. Kimura K, Kataoka S, Ishii Y, Takano T, Yamane K. Nucleotide sequence of the beta-cyclodextrin glucanotransferase gene of alkalophilic Bacillus sp. strain 1011 and similarity of its amino acid sequence to those of alpha-amylases. J Bacteriol 1987;169:4399–402. Kimura K, Ishii Y, Kataoka S, Takano T, Yamane K. Expression of the beta-cyclodextrin glucanotransferase gene of an alkalophilic Bacillus sp. #1011 in Escherichia coli cells and characterization of the synthesized enzyme. Agric Biol Chem 1990;54:641–8. Kitayska T, Petrova P, Ivanova V, Tonkova AI. Purification and properties of a new thermostable cyclodextrin glucanotransferase from Bacillus pseudalcaliphilus 8SB. Appl Biochem Biotechnol 2011;165:1285–95. Klein C, Schulz GE. Structure of cyclodextrin glycosyltransferase refined at 2.0 Å resolution. J Mol Biol 1991;217:737–50. Klein C, Hollender J, Bender H, Schulz GE. Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis. Biochemistry 1992;31:8740–6. Knegtel RM, Strokopytov B, Penninga D, Faber OG, Rozeboom HJ, Kalk KH, et al. Crystallographic studies of the interaction of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 with natural substrates and products. J Biol Chem 1995;270: 29256–64. Knegtel R, Wind RD, Rozeboom HJ, Kalk KH, Buitelaar RM, Dijkhuizen L, et al. Crystal structure at 2.3 Å resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. Order 1996;501:10768. Kumar V. Analysis of the key active subsites of glycoside hydrolase 13 family members. Carbohydr Res 2010;345:893–8. Lawson CL, van Montfort R, Strokopytov B, Rozeboom HJ, Kalk KH, de Vries GE, et al. Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form. J Mol Biol 1994;236:590–600. Lee KW, Shin HD, Lee YH. Catalytic function and affinity purification of site-directed mutant beta-cyclodextrin glucanotransferase from alkalophilic Bacillus furmus var. alkalophilus. Extremophiles. J Mol Catal B Enzym 2003;26:157–65. Lee MH, Yang SJ, Kim JW, Lee HS, Park KH. Characterization of a thermostable cyclodextrin glucanotransferase from Pyrococcus furiosus DSM3638. Extremophiles 2007;11: 537–41. Lee YS, Zhou Y, Park DJ, Chang J, Choi YL. beta-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties. World J Microbiol Biotechnol 2013;29:865–73. Leemhuis H, Uitdehaag JC, Rozeboom HJ, Dijkstra BW, Dijkhuizen L. The remote substrate binding subsite −6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism. J Biol Chem 2002;277:1113–9. Leemhuis H, Rozeboom HJ, Dijkstra BW, Dijkhuizen L. The fully conserved Asp residue in conserved sequence region I of the alpha-amylase family is crucial for the catalytic site architecture and activity. FEBS Lett 2003a;541:47–51. Leemhuis H, Rozeboom HJ, Wilbrink M, Euverink GJ, Dijkstra BW, Dijkhuizen L. Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1. Biochemistry 2003b;42:7518–26. Leemhuis H, Rozeboom HJ, Dijkstra BW, Dijkhuizen L. Improved thermostability of bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge. Proteins 2004;54:128–34. Leemhuis H, Kelly RM, Dijkhuizen L. Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications. Appl Microbiol Biotechnol 2010;85: 823–35. Li D, Roh S-A, Shim J-H, Mikami B, Baik M-Y, Park C-S, et al. Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification. J Agric Food Chem 2005;53:6516–24. Li ZF, Li B, Liu ZG, Wang M, Gu ZB, Du GC, et al. Calcium leads to further increase in glycine-enhanced extracellular secretion of recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli. J Agric Food Chem 2009a;57:6231–7.

R. Han et al. / Biotechnology Advances 32 (2014) 415–428 Li ZF, Zhang JY, Sun Q, Wang M, Gu ZB, Du GC, et al. Mutations of Lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhance beta-cyclodextrin specificity. J Agric Food Chem 2009b;57:8386–91. Li ZF, Zhang JY, Wang M, Gu ZB, Du GC, Li JK, et al. Mutations at subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing alpha-cyclodextrin specificity. Appl Microbiol Biotechnol 2009c;83:483–90. Li ZF, Gu Z, Wang M, Du G, Wu J, Chen J. Delayed supplementation of glycine enhances extracellular secretion of the recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli. Appl Microbiol Biotechnol 2010a;85:553–61. Li ZF, Li B, Gu Z, Du G, Wu J, Chen J. Extracellular expression and biochemical characterization of alpha-cyclodextrin glycosyltransferase from Paenibacillus macerans. Carbohydr Res 2010b;345:886–92. Lin D, Jeang C. Extracellular production of Bacillus macerans authentic cyclodextrin glucanotransferases in Bacillus subtilis. J Chin Agric Chem Soc 1998;36:640–51. Liu H, Li J, Du G, Zhou J, Chen J. Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization. J Ind Microbiol Biotechnol 2012;39:1841–9. Liu L, Xu Q, Han R, Shin HD, Chen RR, Li J, et al. Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans. J Biotechnol 2013;166:198–205. Low KO, Mahadi NM, Abdul Rahim R, Rabu A, Abu Bakar FD, Abdul Murad AM, et al. Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system. J Biotechnol 2010;150:453–9. Low KO, Mahadi NM, Rahim RA, Rabu A, Abu Bakar FD, Murad AM, et al. An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production. J Ind Microbiol Biotechnol 2011;38:1587–97. Martın MT, Angeles Cruces M, Alcalde M, Plou FJ, Bernabé M, Ballesteros A. Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor. Tetrahedron 2004;60:529–34. Nakagawa Y, Takada M, Ogawa K, Hatada Y, Horikoshi K. Site-directed mutations in alanine 223 and glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from alkalophilic Bacillus clarkii 7364 affect cyclodextrin production. J Biochem 2006;140:329–36. Nakamura A, Haga K, Yamane K. Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics. Biochemistry 1994;33:9929–36. Nakano H, Kitahata S, Tominaga Y, Kiku Y, Ando K, Kawashima Y, et al. Syntheses of glucosides with trimethylolpropane and two related polyol moieties by cyclodextrin glucanotransferase and their esterification by lipase. J Ferment Bioeng 1992;73: 237–8. Nakano H, Kiso T, Okamoto K, Tomita T, Manan MB, Kitahata S. Synthesis of glycosyl glycerol by cyclodextrin glucanotransferases. J Biosci Bioeng 2003;95:583–8. Okada S. Method of converting starch to/J-cyclodextrin. 1974; US Patents 3812011. Okada K, Zhao H, Izumi M, Nakajima S, Baba N. Glucosylation of sucrose laurate with cyclodextrin glucanotransferase. Biosci Biotechnol Biochem 2007;71:826–9. Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RNZRA, Illias RM. Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli. J Ind Microbiol Biotechnol 2008;35:1705–14. Paloheimo M, Haglund D, Aho S, Korhola M. Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis. Appl Microbiol Biotechnol 1992;36:584–91. Parsiegla G, Schmidt AK, Schulz GE. Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the gamma-cyclodextrin production. Eur J Biochem 1998;255:710–7. Penninga D, Strokopytov B, Rozeboom HJ, Lawson CL, Dijkstra BW, Bergsma J, et al. Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity. Biochemistry 1995;34:3368–76. Penninga D, van der Veen BA, Knegtel RM, van Hijum SA, Rozeboom HJ, Kalk KH, et al. The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251. J Biol Chem 1996;271:32777–84. Petrova P, Tonkova A, Petrov K. Sequence analysis, cloning and extracellular expression of cyclodextrin glucanotrasferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in E. coli. Process Biochem 2012;47:2139–45. Qi Q, Zimmermann W. Cyclodextrin glucanotransferase: from gene to applications. Appl Microbiol Biotechnol 2005;66:475–85. Radu O, Armand S, Lenouvel F, Driguez H, Cimpean A, Iordachescu D. Luteolin glycosylation in organic solvents under catalytic action of cyclodextrin glycosyltransferase from Bacillus circulans. Rev Roum Chim 2006:147–52. Ramli N, Abd-Aziz S, Alitheen NB, Hassan MA, Maeda T. Improvement of cyclodextrin glycosyltransferase gene expression in Escherichia coli by insertion of regulatory sequences involved in the promotion of RNA transcription. Mol Biotechnol 2013;54:961–8. Rimphanitchayakit V, Tonozuka T, Sakano Y. Construction of chimeric cyclodextrin glucanotransferases from Bacillus circulans A11 and Paenibacillus macerans IAM1243 and analysis of their product specificity. Carbohydr Res 2005;340:2279–89. Rosso A, Ferrarotti S, Krymkiewicz N, Nudel BC. Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R. Microb Cell Factories 2002;1:3. Sato M, Nakamura K, Nagano H, Yagi Y, Koizumi K. Synthesis of glucosyl-inositol using a CGTase, isolation and characterization of the positional isomers, and assimilation profiles for intestinal bacteria. Biotech Lett 1992;14:659–64. Schmid G. Cyclodextrin glycosyltransferase production: yield enhancement by overexpression of cloned genes. Trends Biotechnol 1989;7:244–8.

427

Schmidt AK, Cottaz S, Driguez H, Schulz GE. Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product beta-cyclodextrin. Biochemistry 1998;37:5909–15. Shim JH, Kim YW, Kim TJ, Chae HY, Park JH, Cha H, et al. Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error‐prone PCR. Protein Eng Des Sel 2004;17:205–11. Shim JH, Seo NS, Roh SA, Kim JW, Cha H, Park KH. Improved bread-baking process using Saccharomyces cerevisiae displayed with engineered cyclodextrin glucanotransferase. J Agric Food Chem 2007;55:4735–40. Shimoda K, Hamada H. Production of hesperetin glycosides by Xanthomonas campestris and cyclodextrin glucanotransferase and their anti-allergic activities. Nutrients 2010;2:171–80. Shimoda K, Hara T, Hamada H, Hamada H. Synthesis of curcumin β-maltooligosaccharides through biocatalytic glycosylation with Strophanthus gratus cell culture and cyclodextrin glucanotransferase. Tetrahedron Lett 2007;48:4029–32. Shimoda K, Akagi M, Hamada H. Production of β-maltooligosaccharides of α-and δ-tocopherols by Klebsiella pneumoniae and cyclodextrin glucanotransferase as anti-allergic agents. Molecules 2009;14:3106–14. Sin KA, Nakamura A, Masaki H, Matsuura Y, Uozumi T. Replacement of an amino acid residue of cyclodextrin glucanotransferase of Bacillus ohbensis doubles the production of gamma-cyclodextrin. J Biotechnol 1994;32:283–8. Strokopytov B, Penninga D, Rozeboom HJ, Kalk KH, Dijkhuizen L, Dijkstra BW. X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases. Biochemistry 1995;34:2234–40. Strokopytov B, Knegtel RM, Penninga D, Rozeboom HJ, Kalk KH, Dijkhuizen L, et al. Structure of cyclodextrin glycosyltransferase complexed with a maltononaose inhibitor at 2.6 angstrom resolution. Implications for product specificity. Biochemistry 1996;35: 4241–9. Sugimoto T, Kubota M, Sakai S. New polypeptide with cyclomaltodextrin glucanotransferase activity produced by recombinant DNA techniques. 1986;UK patent application no. GB Patent 2,169,902. Sugimoto K, Nishimura T, Nomura K, Sugimoto K, Kuriki T. Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase. Chem Pharm Bull 2003;51:798–801. Suzuki Y, Suzuki K. Enzymatic formation of 4G-alpha-D-glucopyranosyl-rutin. Agric Biol Chem 1991;55:181–7. Svensson D, Adlercreutz P. Characterisation of a glycosylated alkyl polyglycoside produced by a cyclodextrin glycosyltransferase by HPLC-ELSD and -MS. J Chromatogr B Anal Technol Biomed Life Sci 2011;879:1857–60. Tachibana Y, Takaha T, Fujiwara S, Takagi M, Imanaka T. Acceptor specificity of 4-alpha-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose. J Biosci Bioeng 2000;90:406–9. Takada M, Nakagawa Y, Yamamoto M. Biochemical and genetic analyses of a novel γ-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364. J Biochem 2003;133:317–24. Takano T, Fukuda M, Monma M, Kobayashi S, Kainuma K, Yamane K. Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene. J Bacteriol 1986;166:1118–22. Tesfai BT, Wu D, Chen S, Chen J, Wu J. Strategies for enhancing extracellular secretion of recombinant cyclodextrin glucanotransferase in E. coli. Appl Biochem Biotechnol 2012;167:897–908. Uitdehaag JC, Kalk KH, van Der Veen BA, Dijkhuizen L, Dijkstra BW. The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-Å resolution. J Biol Chem 1999a;274: 34868–76. Uitdehaag JC, Mosi R, Kalk KH, van der Veen BA, Dijkhuizen L, Withers SG, et al. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the alpha-amylase family. Nat Struct Biol 1999b;6:432–6. Uitdehaag JC, van Alebeek GJ, van Der Veen BA, Dijkhuizen L, Dijkstra BW. Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and cyclodextrin size specificity. Biochemistry 2000;39:7772–80. Uitdehaag JC, van der Veen BA, Dijkhuizen L, Elber R, Dijkstra BW. Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase. Proteins 2001;43:327–35. van der Veen BA, Uitdehaag JC, Dijkstra BW, Dijkhuizen L. Engineering of cyclodextrin glycosyltransferase reaction and product specificity. Biochim Biophys Acta 2000a;1543: 336–60. van der Veen BA, Uitdehaag JC, Dijkstra BW, Dijkhuizen L. The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 implications for product inhibition and product specificity. Eur J Biochem 2000b;267:3432–41. van der Veen BA, Uitdehaag JC, Penninga D, van Alebeek GJ, Smith LM, Dijkstra BW, et al. Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production. J Mol Biol 2000c;296:1027–38. van der Veen BA, van Alebeek GJW, Uitdehaag J, Dijkstra BW, Dijkhuizen L. The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms. Eur J Biochem 2000d;267:658–65. van der Veen BA, Leemhuis H, Kralj S, Uitdehaag JC, Dijkstra BW, Dijkhuizen L. Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase. J Biol Chem 2001;276:44557–62. Vetter D, Thorn W, Brunner H, Konig WA. Directed enzymatic synthesis of linear and branched gluco-oligosaccharides, using cyclodextrin-glucanosyltransferase. Carbohydr Res 1992;223:61–9.

428


Wang Z, Qi Q, Wang PG. Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production. Appl Environ Microbiol 2006;72:1873–7. Wang YZ, Feng B, Huang HZ, Kang LP, Cong Y, Zhou WB, et al. Glucosylation of steroidal saponins by cyclodextrin glucanotransferase. Planta Med 2010;76:1724–31. Wang D, Yang L, Fu Z, Xia J. Prediction of thermophilic protein with pseudo amino acid composition: an approach from combined feature selection and reduction. Protein Pept Lett 2011;18:684–9. Wind RD, Uitdehaag JC, Buitelaar RM, Dijkstra BW, Dijkhuizen L. Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1. J Biol Chem 1998;273:5771–9. Yamamoto T, Fujiwara S, Tachibana Y, Takagi M, Fukui K, Imanaka T. Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis. J Biosci Bioeng 2000;89:206–9. Yenpetch W, Packdibamrung K, Zimmermann W, Pongsawasdi P. Evidence of the involvement of asparagine deamidation in the formation of cyclodextrin glycosyltransferase isoforms in Paenibacillus sp. RB01. Mol Biotechnol 2011;47:234–42.

Yoon SH, Robyt JF. Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase. Carbohydr Res 2006;341:210–7. Yoon S-H, Bruce Fulton D, Robyt JF. Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase. Carbohydr Res 2004;339: 1517–29. Yoshinaga K, Abe J-i, Tanimoto T, Koizumi K, Hizukuri S. Preparation and reactivity of a novel disaccharide, glucosyl 1, 5-anhydro-d-fructose (1, 5-anhydro-3-O-α-glucopyranosyld-fructose). Carbohydr Res 2003;338:2221–5. Zhang ZC, Li JH, Liu L, Sun J, Hua ZZ, Du GC, et al. Enzymatic transformation of 2-O-alphaD-glucopyranosyl-L-ascorbic acid by alpha-cyclodextrin glucanotransferase from recombinant Escherichia coli. Biotechnol Bioproc Eng 2011;16:107–13. Zheng M, Endo T, Zimmermann W. Enzymatic synthesis and analysis of large-ring cyclodextrins. Aust J Chem 2002;55:39–48. Zhou J, Liu H, Du G, Li J, Chen J. Production of α-cyclodextrin glycosyltransferase in Bacillus megaterium MS941 by systematic codon usage optimization. J Agric Food Chem 2012;60:10285–92.

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