FEMS Microbiology Letters, 362, 2015, fnv054 doi: 10.1093/femsle/fnv054 Advance Access Publication Date: 2 April 2015 Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution Shangyong Li† , Xuemei Yang† , Mengmeng Bao, Ying Wu, Wengong Yu and Feng Han∗ Key Laboratory of Marine Drugs, Chinese Ministry of Education; Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology; School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China ∗ Corresponding author: School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. Tel: +86-532-82032067;
Fax: +86-532-82033054; E-mail: [email protected] These authors contributed equally to this work. One sentence summary: This is the first report of the function of the carbohydrate-binding module in alginate lyase Editor: Michael Sauer †
ABSTRACT The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7–6.6 times those of AlyL2-CM at 30–50◦ C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms. Keywords: alginate lyase; carbohydrate-binding module; catalytic activity; thermostability; product distribution
INTRODUCTION Alginate, an acidic heteropolysaccharide, consists of β-dmannuronate (M) and α-l-guluronate (G) which are arranged into polyM, polyG and an alternating or random polyMG block (Falkeborga, Cheonga, Gianficoa et al. 2014). Alginate is the most
abundant carbohydrate in brown algae and is widely used in the food and pharmaceutical industries due to its high viscosity and gelling property. Recently, the application of alginate for bioethanol production has received increasing attention (Wargacki, Leonard, Win et al. 2012; Enquist-Newman, Faust, Bravo et al. 2014).
Received: 4 March 2015; Accepted: 29 March 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2015, Vol. 362, No. 10
Alginate lyases are enzymes that catalyze the degradation of alginate with a β-elimination mechanism that breaks the 1–4 O-linkages between the uronic acids in the linear polymer (Kim, Lee and Lee 2011). The enzymatic degradation products have been found to exhibit a variety of bioactive functions such as bifidobacteria growth-improving activities (Wang, Han, Hu et al. 2006), cytokine-inducing activity in mononuclear cells (Iwamoto, Xu, Tamura et al. 2003), antioxidant activity (Falkeborga, Cheonga, Gianficoa et al. 2014), endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress-protecting activities (Tusi, Khalaj, Ashabi et al. 2011) and plant root growth-promoting activities (Iwasaki and Matsubara 2000). Alginate lyases have been used in the production of alginate oligosaccharides, in the protoplast production of brown algae, in the elucidation of alginate structure and in the application of cystic fibrosis management (Alkawash, Soothill and Schiller 2006; Alipour, Suntres and Omri 2009; Høiby, Bjarnsholt, Givskov et al. 2010; Inoue, Mashino, Kodama et al. 2011). Several thousands alginate lyase-encoding genes have been found from marine microorganisms, brown seaweeds and mollusks, and grouped under polysaccharide lyase (PL) families 5, 6, 7, 14, 15, 17 and 18 in the CAZy database (http://www.cazy.org/). According to the sequence information, some alginate lyases are modular proteins containing a catalytic module and one or more carbohydrate-binding modules (CBMs) (Sawabe, Takahashi, Ezura et al. 2001; Duan, Han and Yu 2009). The most abundant CBMs in alginate lyase are CBM13, CBM16 and CBM32. However, the function of these CBMs in alginate lyases has not been elucidated. In this study, a new alginate lyase gene, alyL2, whose product contains an N-terminal CBM13 module and a C-terminal PL7 catalytic module, was cloned from Agarivorans sp. L11. The fulllength AlyL2-FL and its catalytic module, AlyL2-CM, were overexpressed in Escherichia coli and characterized. It turned out that the CBM13 module could clearly improve the catalytic efficiency and thermostability; it also altered the product specificity of alginate lyase AlyL2.
MATERIALS AND METHODS Bacterial strains, plasmids and oligonucleotides Agarivorans sp. L11 was isolated from the coastal zone of Jiaozhou Bay, Qingdao, China as described before (Li, Yang, Zhang et al. 2014). Escherichia coli strains DH5α and BL21(DE3) (Novagen) were grown at 37◦ C in Luria–Bertani (LB) broth or on LB agar supplemented with kanamycin (30 μg/ml) when relevant. Oligonucleotides used for the gene cloning and expression are shown in Supplementary Table S1.
5.1.zip). The conserved domains of AlyL2 were searched with InterProScan 4 running the HMMPfam application (http://www. ebi.ac.uk/Tools/pfa/iprscan/). Multiple sequence alignment was performed with the ClustalX program. The theoretical isoelectric point (pI) and molecular weight (Mw) were calculated using the Compute pI/Mw Tool (http://web.expasy.org/compute pi/).
Expression and purification of recombinant AlyL2-FL and AlyL2-CM To express the full-length AlyL2 (AlyL2-FL) and its truncated mutant (catalytic module only, AlyL2-CM), the appropriate DNA fragments were amplified using the primers (PalyL2-FL-F/R and PalyL2-CM-F/R), digested with NdeI and XhoI, then ligated into the similarly digested plasmid pET-28a(+). Protein expression was induced at an OD600 of 0.4 with 0.05 mM isopropyl-βthiogalactopyranoside (IPTG) for 40 h at 18◦ C and 100 rpm. Cells were harvested and disrupted by sonication, then cell debris and unbroken cells were removed by centrifugation. The recombinant AlyL2-FL and AlyL2-CM were purified from the soluble fraction using an Ni-Sepharose column. The molecular weights of purified proteins were determined by SDS–PAGE on a 10% (w/v) resolving gel.
Assay of alginate lyase activity The alginate lyase-catalyzed ‘β-elimination’ reaction was conducted with 100 μl of enzyme solution and 900 μl of substrate solution [0.3% (w/v) alginate, 20 mM phosphate buffer plus 500 mM NaCl, pH 7.0] at 40◦ C for 10 min. One unit (U) was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.1 per min.
Biochemical characterization of AlyL2-FL and AlyL2-CM The optimal pH of the enzymatic activity was determined using 50 mM Na2 HPO4 –citric acid (pH 3.0–6.0), 50 mM Na2 HPO4 – NaH2 PO4 (pH 6.0–8.0) and 50 mM glycine-NaOH (pH 8.0–10.6) buffers in the assay system. To evaluate pH stability, the residual activity was measured after the enzymes were incubated in the above buffers for 6 h at 4◦ C. The optimal temperature of the enzymatic activity was determined by measuring the activity at various temperatures (10–60◦ C). The thermostability of the enzymes was studied by measuring the residual activity after the enzymes were incubated at 0–60◦ C for 1 h in 20 mM phosphate buffer (pH 7.0). The half-lives (t1/2 ), the time point at which the residual activity is 50% of the initial alginate lyase activity, was determined at 10–50◦ C. The effects of metal ions and chelators on AlyL2-FL and AlyL2-CM activity were examined by monitoring enzymatic activity in the presence of various cations or chelators.
Cloning and sequence analysis of the alginate lyase gene
Assay of kinetic parameters and substrate specificity
Degenerate primers (PalyL2-F1 and PalyL2-R1) were designed according to the conserved amino acid sequences of the alginate lyases in the PL-7 family; the partial sequence of the alyL2 gene (650 bp in length) was obtained and sequenced. The flanking sequences were obtained using the SiteFinding-PCR method with nested specific primers (SFP1&2&3, PalyL2-down-sF1&2&3, PalyL2-up-sF1&2&3). The nucleotide sequence for alyL2 was deposited in GenBank under the accession number KP306765. The open reading frame (ORF) was identified with the DNATools program ORF finder (ftp://sgst ftp.scbit.org/software/DNATools
The kinetic parameters of AlyL2-FL and AlyL2-CM were measured by using 10 different concentrations of alginate (ranging from 0.1 to 8 mg/ml) in 20 mM phosphate buffer plus 0.5 M NaCl (pH 7.0) at 40◦ C for 5 min, followed by the addition of the enzyme to obtain a final protein concentration of 8.1 nM. The Km and catalytic efficient (kcat /Km ) values were determined with the Hyper32 program (Swift, Hudgens, Heselpoth et al. 2014). For the studies of substrate specificities, sodium alginate, polyM and polyG [0.3% (w/v) in 20 mM phosphate buffer plus 500 mM NaCl, pH 7.0] were used as the substrate in the standard enzymatic
Li et al.
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assay described previously. PolyM and polyG (purity: ∼95%) were kindly provided by Professors Guangli Yu and Chunxia Li (School of Medicine and Pharmacy, Ocean University of China, China).
Alginate binding assay The strength of binding of AlyL2-CBM13 to soluble alginate was evaluated by a native affinity electrophoresis method as described previously (Cheng, Hong, Liu et al. 2009), in which 0.15% (w/v) alginate was included in the native polyacrylamide gel. Bovine serum albumin (BSA) was used as the control protein.
Analysis of reaction pattern and products of AlyL2-FL and AlyL2-CM To determine the action pattern of AlyL2-FL and AlyL2-CM, the enzymes (0.5 mg) were added to 10 ml of sodium alginate (1 mg/ml) and incubated at 40◦ C for 1, 5, 15 or 30 min. The reaction products were analyzed using a fast protein liquid chromatography (FPLC) with a superdex peptide 10/300 gel filtration column (GE Health, USA) monitored at 235 nm, in which the mobile phase was 0.2 M ammonium bicarbonate at a flow rate of 0.3 ml/min. The ratio of degradation products was analyzed by the peak integrates of the UNICORN 5.31. The molecular masses of the main products obtained from the reaction of alginate for 30 min with AlyL2-FL and AlyL2-CM were determined using negative-ion electrospray ionization mass spectrometry (ESI-MS). Then the fractions of end-product were separated by a Biogel-P6 column (Biorad, USA) and analyzed by 1 H-nuclear magnetic resonance (NMR) spectrometry.
RESULTS AND DISCUSSION Cloning and sequence analysis of the alginate lyase gene The alginate lyase gene, alyL2, consisted of an ORF of 1335 bp, encoding 444 amino acid residues. AlyL2 had the highest identity of 98% with a hypothetical alginate lyase precursor (WP 016402785) from Agarivorans albus. So far, there are only three alginate lyases (A1m, AlmU and AlyL1) that have been characterized from the genus Agarivorans (Kobayashi, Uchimura, Miyazaki et al. 2009; Uchimura, Miyazaki, Nogi et al. 2010; Li, Yang, Zhang et al. 2014). AlyL2 showed only 26.5, 11.8 and 24.5% identities with Alm (286 amino acids), AlmU (309 amino acids) and AlyL1 (349 amino acids), respectively. AlyL2 possessed a CBM (Thr6–Trp133) at the N-terminus and a PL-7 catalytic domain (Phe167–His439) at the C-terminus (Supplementary Fig. S1). The molecular weight and pI of the full-length enzyme (AlyL2-FL), the catalytic domain (AlyL2-CM) and the carbohydrate-binding module (AlyL2-CBM) deduced from their amino acid sequences were 48 086, 33 019 and 17 002 Da and 4.95, 4.66 and 6.70, respectively. AlyL2-CBM had the highest identity of 87.6% with a CBM13 module in βagarase (ABW77762). CBM13 was also found in a number of glycoside hydrolases (GHs) and PLs (Fujimoto 2013). In xylanase, CBM13 could improve the catalytic efficiency of enzymes by increasing their local concentrations around the insoluble xylan ´ Correia, Romao ˜ et al. 2011; Fujimoto 2013). However, the (Bras, function of CBM13 in alginate lyase has not been characterized.
Expression and purification of AlyL2-FL and AlyL2-CM The full-length gene, alyL2-FL, and its truncated mutant, alyL2CM, were expressed in the E. coli BL21(DE3)/pET-28a(+) system.
Figure 1. SDS–PAGE of the purified alginate lyase. Lane M, marker; lane 1, purified AlyL2-FL; lane 2, purified AlyL2-CM.
The recombinant AlyL2-FL and AlyL2-CM were purified to apparent homogeneity, and the overall yields were 71.9% and 67.2%, respectively. The molecular masses of the recombinant AlyL2FL and AlyL2-CM were estimated to be ∼50 and 34 kDa by SDS– PAGE (Fig. 1), which were in good agreement with their theoretical molecular masses. The specific activity of purified AlyL2-FL was 81 220.4 U/μmol, which was much higher than that of the purified AlyL2-CM (39 622.3 U/μmol). As documented previously, CBM13 could improve the catalytic efficiency towards the insoluble substrate in xylanases; however, it was not essential for hydrolysis of soluble xylan (Notenboom, Boraston, Williams et al. ¨ ¨ Fagerstrom ¨ et al. 2005; Fujimoto 2013). 2002; Leskinen. Mantyl a, To the best of our knowledge, this is the first report showing that CBM13 could enhance the catalytic efficiency towards the soluble substrate.
Biochemical characterization of AlyL2-FL and AlyL2-CM When assayed at various pH values, the maximum activities of AlyL2-FL and AlyL2-CM were observed at pH 8.6 and 7.0, respectively (Fig. 2A). AlyL2-FL retained >80% of its initial activity after incubation at pH 3.0–10.0 for 6 h. However, no more than 30% of AlyL2-CM activity was retained in the pH range of 3.0–6.0 (Fig. 2B). These results indicated that AlyL2-FL is more stable than AlyL2-CM over a broader pH range. The optimal temperatures of AlyL2-FL and AlyL2-CM were 45 and 35◦ C, respectively (Fig. 2C). Only 40% and 10% of AlyL2CM activity remained after it was incubated at 30 and 40◦ C for 1 h, respectively; while, AlyL2-FL retained 95% and 80% of activity, respectively, under the same conditions (Fig. 2D). The halflives (t1/2 ) of the enzymes at various temperatures were also determined. At 10 and 20◦ C, no reduction in enzyme activity
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Figure 2. Effects of pH and temperature on the activities and stabilities of AlyL2-FL (solid line) and AlyL2-CM (dotted line). (A) The optimal pH values of AlyL2-FL and AlyL2-CM were determined by measuring the activity in 50 mM Na2 HPO4 –citric acid (filled diamonds), 50 mM Na2 HPO4 –NaH2 PO4 (open triangles) and 50 mM Gly-NaOH (filled circles) buffers. (B) The pH stabilities of AlyL2-FL and AlyL2-CM. The residual activity was measured after the enzyme was incubated in the pH range of 3.0–10.6 with the above buffers for 6 h at 4◦ C. (C) The optimal temperatures of the enzymes were determined by measuring the activity at various temperatures (10–60◦ C). (D) The thermostabilities of AlyL2-FL and AlyL2-CM. The residual activity was determined at the optimal temperatures after incubation at various temperatures (0–60◦ C) for 1 h.
Table 1. The half-lives (t1/2 ) of recombinant AlyL2-FL and AlyL2-CM. t1/2 (min)a Enzyme AlyL2-FL AlyL2-CM
30o C
40o C
50o C
365 ± 5 55 ± 5
125 ± 10 25 ± 5
70 ± 5 15 ± 5
Table 2. The substrate specificity of AlyL2-FL and AlyL2-CM. Substrate
AlyL2-FL (%)
AlyL2-CM (%)
Alginate PolyM PolyG
100 ± 1.6a 121.8 ± 4.9 40.5 ± 3.2
100 ± 2.5a 165.5 ± 3.6 138.8 ± 4.4
a a
Time point at which the residual activity is 50% of the initial alginate lyase activity.
was found even after 6 h of incubation (data not shown). At 30, 40 and 50◦ C, the half-lives of AlyL2-FL were ∼4.7–6.6 times those of AlyL2-CM (Table 1). CBM13 has not been known as a thermostability-improving domain in previous studies. In xylanase, the C-terminal CBM13 decreased the thermal stabil¨ ¨ Fagerstrom ¨ ity of XynB (Leskinen. Mantyl a, et al. 2005). Alginate lyases from the genus Pseudoalteromonas (Sawabe, Takahashi, Ezura et al. 2001; Duan, Han and Yu 2009), Agarivorans (Kobayashi, Uchimura, Miyasaki et al. 2009; Li, Yang, Zhang et al. 2014), Microbulbifer (Swift, Hudgens, Heselpoth et al. 2014) and most of Vibrio (Wong, Preston and Schiller 2000; Uchimura, Miyazaki, Nogi et al. 2010) were stable only at temperatures below 40◦ C. Alginate lyase, AlyV5, from Vibrio sp. QY105 and alginate lyase from Isoptericola halotolerans CGMCC 5336 retained >75% of their activity after incubation at temperatures
R E S E A R C H L E T T E R – Physiology & Biochemistry
Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution Shangyong Li† , Xuemei Yang† , Mengmeng Bao, Ying Wu, Wengong Yu and Feng Han∗ Key Laboratory of Marine Drugs, Chinese Ministry of Education; Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology; School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China ∗ Corresponding author: School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. Tel: +86-532-82032067;
Fax: +86-532-82033054; E-mail: [email protected] These authors contributed equally to this work. One sentence summary: This is the first report of the function of the carbohydrate-binding module in alginate lyase Editor: Michael Sauer †
ABSTRACT The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7–6.6 times those of AlyL2-CM at 30–50◦ C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms. Keywords: alginate lyase; carbohydrate-binding module; catalytic activity; thermostability; product distribution
INTRODUCTION Alginate, an acidic heteropolysaccharide, consists of β-dmannuronate (M) and α-l-guluronate (G) which are arranged into polyM, polyG and an alternating or random polyMG block (Falkeborga, Cheonga, Gianficoa et al. 2014). Alginate is the most
abundant carbohydrate in brown algae and is widely used in the food and pharmaceutical industries due to its high viscosity and gelling property. Recently, the application of alginate for bioethanol production has received increasing attention (Wargacki, Leonard, Win et al. 2012; Enquist-Newman, Faust, Bravo et al. 2014).
Received: 4 March 2015; Accepted: 29 March 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2015, Vol. 362, No. 10
Alginate lyases are enzymes that catalyze the degradation of alginate with a β-elimination mechanism that breaks the 1–4 O-linkages between the uronic acids in the linear polymer (Kim, Lee and Lee 2011). The enzymatic degradation products have been found to exhibit a variety of bioactive functions such as bifidobacteria growth-improving activities (Wang, Han, Hu et al. 2006), cytokine-inducing activity in mononuclear cells (Iwamoto, Xu, Tamura et al. 2003), antioxidant activity (Falkeborga, Cheonga, Gianficoa et al. 2014), endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress-protecting activities (Tusi, Khalaj, Ashabi et al. 2011) and plant root growth-promoting activities (Iwasaki and Matsubara 2000). Alginate lyases have been used in the production of alginate oligosaccharides, in the protoplast production of brown algae, in the elucidation of alginate structure and in the application of cystic fibrosis management (Alkawash, Soothill and Schiller 2006; Alipour, Suntres and Omri 2009; Høiby, Bjarnsholt, Givskov et al. 2010; Inoue, Mashino, Kodama et al. 2011). Several thousands alginate lyase-encoding genes have been found from marine microorganisms, brown seaweeds and mollusks, and grouped under polysaccharide lyase (PL) families 5, 6, 7, 14, 15, 17 and 18 in the CAZy database (http://www.cazy.org/). According to the sequence information, some alginate lyases are modular proteins containing a catalytic module and one or more carbohydrate-binding modules (CBMs) (Sawabe, Takahashi, Ezura et al. 2001; Duan, Han and Yu 2009). The most abundant CBMs in alginate lyase are CBM13, CBM16 and CBM32. However, the function of these CBMs in alginate lyases has not been elucidated. In this study, a new alginate lyase gene, alyL2, whose product contains an N-terminal CBM13 module and a C-terminal PL7 catalytic module, was cloned from Agarivorans sp. L11. The fulllength AlyL2-FL and its catalytic module, AlyL2-CM, were overexpressed in Escherichia coli and characterized. It turned out that the CBM13 module could clearly improve the catalytic efficiency and thermostability; it also altered the product specificity of alginate lyase AlyL2.
MATERIALS AND METHODS Bacterial strains, plasmids and oligonucleotides Agarivorans sp. L11 was isolated from the coastal zone of Jiaozhou Bay, Qingdao, China as described before (Li, Yang, Zhang et al. 2014). Escherichia coli strains DH5α and BL21(DE3) (Novagen) were grown at 37◦ C in Luria–Bertani (LB) broth or on LB agar supplemented with kanamycin (30 μg/ml) when relevant. Oligonucleotides used for the gene cloning and expression are shown in Supplementary Table S1.
5.1.zip). The conserved domains of AlyL2 were searched with InterProScan 4 running the HMMPfam application (http://www. ebi.ac.uk/Tools/pfa/iprscan/). Multiple sequence alignment was performed with the ClustalX program. The theoretical isoelectric point (pI) and molecular weight (Mw) were calculated using the Compute pI/Mw Tool (http://web.expasy.org/compute pi/).
Expression and purification of recombinant AlyL2-FL and AlyL2-CM To express the full-length AlyL2 (AlyL2-FL) and its truncated mutant (catalytic module only, AlyL2-CM), the appropriate DNA fragments were amplified using the primers (PalyL2-FL-F/R and PalyL2-CM-F/R), digested with NdeI and XhoI, then ligated into the similarly digested plasmid pET-28a(+). Protein expression was induced at an OD600 of 0.4 with 0.05 mM isopropyl-βthiogalactopyranoside (IPTG) for 40 h at 18◦ C and 100 rpm. Cells were harvested and disrupted by sonication, then cell debris and unbroken cells were removed by centrifugation. The recombinant AlyL2-FL and AlyL2-CM were purified from the soluble fraction using an Ni-Sepharose column. The molecular weights of purified proteins were determined by SDS–PAGE on a 10% (w/v) resolving gel.
Assay of alginate lyase activity The alginate lyase-catalyzed ‘β-elimination’ reaction was conducted with 100 μl of enzyme solution and 900 μl of substrate solution [0.3% (w/v) alginate, 20 mM phosphate buffer plus 500 mM NaCl, pH 7.0] at 40◦ C for 10 min. One unit (U) was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.1 per min.
Biochemical characterization of AlyL2-FL and AlyL2-CM The optimal pH of the enzymatic activity was determined using 50 mM Na2 HPO4 –citric acid (pH 3.0–6.0), 50 mM Na2 HPO4 – NaH2 PO4 (pH 6.0–8.0) and 50 mM glycine-NaOH (pH 8.0–10.6) buffers in the assay system. To evaluate pH stability, the residual activity was measured after the enzymes were incubated in the above buffers for 6 h at 4◦ C. The optimal temperature of the enzymatic activity was determined by measuring the activity at various temperatures (10–60◦ C). The thermostability of the enzymes was studied by measuring the residual activity after the enzymes were incubated at 0–60◦ C for 1 h in 20 mM phosphate buffer (pH 7.0). The half-lives (t1/2 ), the time point at which the residual activity is 50% of the initial alginate lyase activity, was determined at 10–50◦ C. The effects of metal ions and chelators on AlyL2-FL and AlyL2-CM activity were examined by monitoring enzymatic activity in the presence of various cations or chelators.
Cloning and sequence analysis of the alginate lyase gene
Assay of kinetic parameters and substrate specificity
Degenerate primers (PalyL2-F1 and PalyL2-R1) were designed according to the conserved amino acid sequences of the alginate lyases in the PL-7 family; the partial sequence of the alyL2 gene (650 bp in length) was obtained and sequenced. The flanking sequences were obtained using the SiteFinding-PCR method with nested specific primers (SFP1&2&3, PalyL2-down-sF1&2&3, PalyL2-up-sF1&2&3). The nucleotide sequence for alyL2 was deposited in GenBank under the accession number KP306765. The open reading frame (ORF) was identified with the DNATools program ORF finder (ftp://sgst ftp.scbit.org/software/DNATools
The kinetic parameters of AlyL2-FL and AlyL2-CM were measured by using 10 different concentrations of alginate (ranging from 0.1 to 8 mg/ml) in 20 mM phosphate buffer plus 0.5 M NaCl (pH 7.0) at 40◦ C for 5 min, followed by the addition of the enzyme to obtain a final protein concentration of 8.1 nM. The Km and catalytic efficient (kcat /Km ) values were determined with the Hyper32 program (Swift, Hudgens, Heselpoth et al. 2014). For the studies of substrate specificities, sodium alginate, polyM and polyG [0.3% (w/v) in 20 mM phosphate buffer plus 500 mM NaCl, pH 7.0] were used as the substrate in the standard enzymatic
Li et al.
3
assay described previously. PolyM and polyG (purity: ∼95%) were kindly provided by Professors Guangli Yu and Chunxia Li (School of Medicine and Pharmacy, Ocean University of China, China).
Alginate binding assay The strength of binding of AlyL2-CBM13 to soluble alginate was evaluated by a native affinity electrophoresis method as described previously (Cheng, Hong, Liu et al. 2009), in which 0.15% (w/v) alginate was included in the native polyacrylamide gel. Bovine serum albumin (BSA) was used as the control protein.
Analysis of reaction pattern and products of AlyL2-FL and AlyL2-CM To determine the action pattern of AlyL2-FL and AlyL2-CM, the enzymes (0.5 mg) were added to 10 ml of sodium alginate (1 mg/ml) and incubated at 40◦ C for 1, 5, 15 or 30 min. The reaction products were analyzed using a fast protein liquid chromatography (FPLC) with a superdex peptide 10/300 gel filtration column (GE Health, USA) monitored at 235 nm, in which the mobile phase was 0.2 M ammonium bicarbonate at a flow rate of 0.3 ml/min. The ratio of degradation products was analyzed by the peak integrates of the UNICORN 5.31. The molecular masses of the main products obtained from the reaction of alginate for 30 min with AlyL2-FL and AlyL2-CM were determined using negative-ion electrospray ionization mass spectrometry (ESI-MS). Then the fractions of end-product were separated by a Biogel-P6 column (Biorad, USA) and analyzed by 1 H-nuclear magnetic resonance (NMR) spectrometry.
RESULTS AND DISCUSSION Cloning and sequence analysis of the alginate lyase gene The alginate lyase gene, alyL2, consisted of an ORF of 1335 bp, encoding 444 amino acid residues. AlyL2 had the highest identity of 98% with a hypothetical alginate lyase precursor (WP 016402785) from Agarivorans albus. So far, there are only three alginate lyases (A1m, AlmU and AlyL1) that have been characterized from the genus Agarivorans (Kobayashi, Uchimura, Miyazaki et al. 2009; Uchimura, Miyazaki, Nogi et al. 2010; Li, Yang, Zhang et al. 2014). AlyL2 showed only 26.5, 11.8 and 24.5% identities with Alm (286 amino acids), AlmU (309 amino acids) and AlyL1 (349 amino acids), respectively. AlyL2 possessed a CBM (Thr6–Trp133) at the N-terminus and a PL-7 catalytic domain (Phe167–His439) at the C-terminus (Supplementary Fig. S1). The molecular weight and pI of the full-length enzyme (AlyL2-FL), the catalytic domain (AlyL2-CM) and the carbohydrate-binding module (AlyL2-CBM) deduced from their amino acid sequences were 48 086, 33 019 and 17 002 Da and 4.95, 4.66 and 6.70, respectively. AlyL2-CBM had the highest identity of 87.6% with a CBM13 module in βagarase (ABW77762). CBM13 was also found in a number of glycoside hydrolases (GHs) and PLs (Fujimoto 2013). In xylanase, CBM13 could improve the catalytic efficiency of enzymes by increasing their local concentrations around the insoluble xylan ´ Correia, Romao ˜ et al. 2011; Fujimoto 2013). However, the (Bras, function of CBM13 in alginate lyase has not been characterized.
Expression and purification of AlyL2-FL and AlyL2-CM The full-length gene, alyL2-FL, and its truncated mutant, alyL2CM, were expressed in the E. coli BL21(DE3)/pET-28a(+) system.
Figure 1. SDS–PAGE of the purified alginate lyase. Lane M, marker; lane 1, purified AlyL2-FL; lane 2, purified AlyL2-CM.
The recombinant AlyL2-FL and AlyL2-CM were purified to apparent homogeneity, and the overall yields were 71.9% and 67.2%, respectively. The molecular masses of the recombinant AlyL2FL and AlyL2-CM were estimated to be ∼50 and 34 kDa by SDS– PAGE (Fig. 1), which were in good agreement with their theoretical molecular masses. The specific activity of purified AlyL2-FL was 81 220.4 U/μmol, which was much higher than that of the purified AlyL2-CM (39 622.3 U/μmol). As documented previously, CBM13 could improve the catalytic efficiency towards the insoluble substrate in xylanases; however, it was not essential for hydrolysis of soluble xylan (Notenboom, Boraston, Williams et al. ¨ ¨ Fagerstrom ¨ et al. 2005; Fujimoto 2013). 2002; Leskinen. Mantyl a, To the best of our knowledge, this is the first report showing that CBM13 could enhance the catalytic efficiency towards the soluble substrate.
Biochemical characterization of AlyL2-FL and AlyL2-CM When assayed at various pH values, the maximum activities of AlyL2-FL and AlyL2-CM were observed at pH 8.6 and 7.0, respectively (Fig. 2A). AlyL2-FL retained >80% of its initial activity after incubation at pH 3.0–10.0 for 6 h. However, no more than 30% of AlyL2-CM activity was retained in the pH range of 3.0–6.0 (Fig. 2B). These results indicated that AlyL2-FL is more stable than AlyL2-CM over a broader pH range. The optimal temperatures of AlyL2-FL and AlyL2-CM were 45 and 35◦ C, respectively (Fig. 2C). Only 40% and 10% of AlyL2CM activity remained after it was incubated at 30 and 40◦ C for 1 h, respectively; while, AlyL2-FL retained 95% and 80% of activity, respectively, under the same conditions (Fig. 2D). The halflives (t1/2 ) of the enzymes at various temperatures were also determined. At 10 and 20◦ C, no reduction in enzyme activity
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FEMS Microbiology Letters, 2015, Vol. 362, No. 10
Figure 2. Effects of pH and temperature on the activities and stabilities of AlyL2-FL (solid line) and AlyL2-CM (dotted line). (A) The optimal pH values of AlyL2-FL and AlyL2-CM were determined by measuring the activity in 50 mM Na2 HPO4 –citric acid (filled diamonds), 50 mM Na2 HPO4 –NaH2 PO4 (open triangles) and 50 mM Gly-NaOH (filled circles) buffers. (B) The pH stabilities of AlyL2-FL and AlyL2-CM. The residual activity was measured after the enzyme was incubated in the pH range of 3.0–10.6 with the above buffers for 6 h at 4◦ C. (C) The optimal temperatures of the enzymes were determined by measuring the activity at various temperatures (10–60◦ C). (D) The thermostabilities of AlyL2-FL and AlyL2-CM. The residual activity was determined at the optimal temperatures after incubation at various temperatures (0–60◦ C) for 1 h.
Table 1. The half-lives (t1/2 ) of recombinant AlyL2-FL and AlyL2-CM. t1/2 (min)a Enzyme AlyL2-FL AlyL2-CM
30o C
40o C
50o C
365 ± 5 55 ± 5
125 ± 10 25 ± 5
70 ± 5 15 ± 5
Table 2. The substrate specificity of AlyL2-FL and AlyL2-CM. Substrate
AlyL2-FL (%)
AlyL2-CM (%)
Alginate PolyM PolyG
100 ± 1.6a 121.8 ± 4.9 40.5 ± 3.2
100 ± 2.5a 165.5 ± 3.6 138.8 ± 4.4
a a
Time point at which the residual activity is 50% of the initial alginate lyase activity.
was found even after 6 h of incubation (data not shown). At 30, 40 and 50◦ C, the half-lives of AlyL2-FL were ∼4.7–6.6 times those of AlyL2-CM (Table 1). CBM13 has not been known as a thermostability-improving domain in previous studies. In xylanase, the C-terminal CBM13 decreased the thermal stabil¨ ¨ Fagerstrom ¨ ity of XynB (Leskinen. Mantyl a, et al. 2005). Alginate lyases from the genus Pseudoalteromonas (Sawabe, Takahashi, Ezura et al. 2001; Duan, Han and Yu 2009), Agarivorans (Kobayashi, Uchimura, Miyasaki et al. 2009; Li, Yang, Zhang et al. 2014), Microbulbifer (Swift, Hudgens, Heselpoth et al. 2014) and most of Vibrio (Wong, Preston and Schiller 2000; Uchimura, Miyazaki, Nogi et al. 2010) were stable only at temperatures below 40◦ C. Alginate lyase, AlyV5, from Vibrio sp. QY105 and alginate lyase from Isoptericola halotolerans CGMCC 5336 retained >75% of their activity after incubation at temperatures
