Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1188-3
ORIGINAL PAPER
Optimal production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases Da Mao Wang • Hee Taek Kim • Eun Ju Yun Do Hyoung Kim • Yong-Cheol Park • Hee Chul Woo • Kyoung Heon Kim
•
Received: 9 October 2013 / Accepted: 6 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of
D. M. Wang E. J. Yun D. H. Kim K. H. Kim (&) Department of Biotechnology, Korea University Graduate School, Seoul 136-713, Republic of Korea e-mail: [email protected] H. T. Kim Research Center for Biobased Chemistry, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea Y.-C. Park Department of Bio and Fermentation Convergence, Kookmin University, Seoul 136-702, Republic of Korea H. C. Woo Department of Chemical Engineering, Pukyung National University, Busan 608-739, Republic of Korea
DEH that is the key substrate for producing biofuels from brown macro algal biomass. Keywords Alginate lyase Brown macro algae Uronic acid Saccharification Saccharophagus degradans
Introduction Biofuel production using first-generation biomass such as sugar and starch can lead to conflicts with food production due to the increasing worldwide food consumption [1]. Alternatively, second-generation biomass represented by lignocellulose is actively considered, but it requires substantial pretreatment steps [2]. Algal biomass has been gaining importance as a third-generation biofuel resource for the production of fuels and chemicals [3]. In particular, macroalgae, which contain a large amount of carbohydrates, can be utilized as sources of fermentable sugars [4–6]. The dominant carbohydrate in brown macroalgae is alginate, which comprises 12–34 % of its total dry mass [7]. Alginate is a linear polysaccharide composed of b-D-mannuronic acid (M) and its C5 epimer, a-L-guluronic acid (G) [8]. An important prerequisite step for alginate metabolism by microorganisms is its depolymerization into sugars with low degrees of polymerization (DP), such as oligosaccharides and monosaccharides. The most commonly employed processes for alginate depolymerization are thermochemical and biochemical conversions. Thermochemical conversions are single-step processes that consist of hydrothermal treatments [9, 10] with catalysts, such as oxalic acid [11], which result in the formation of oligomers as well as byproducts, such as lactic acid, glycolic acid, and other water-soluble substances.
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Bioprocess Biosyst Eng Fig. 1 Schematic diagram for the enzymatic depolymerization of alginate using endo- and exotype alginate lyases
In contrast, biochemical depolymerization of alginate is a multi-step process that includes enzymatic and nonenzymatic conversions. The enzymatic depolymerization process is carried out using alginate lyases (Fig. 1). These lyases can be classified into two major groups based on the mode of degradation of alginate: endo-type alginate lyases cleave within the chain and produce unsaturated uronic acid oligomers with a double bond between C4 and C5 at the nonreducing end, whereas exo-type alginate lyases cleave at the termini of the chains to produce monosaccharides from oligosaccharides. The monomers are then nonenzymatically converted into an a-ketouronate, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) [12]. DEH is a key starting material, which can be converted into 2-keto-3-deoxy-D-gluconate (KDG) by a reductase. KDG, another important intermediate, enters the Entner–Doudoroff pathway to generate building blocks and energy for cellular metabolism [13]. DEH is the most abundant substrate obtainable from brown macroalgae, and this sugar acid that is not fermented by general microorganisms is now becoming convertible into ethanol or other chemicals using recombinant microorganisms [14–16]. Therefore, DEH is the key player in the fuel production using brown macro algal
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biomass, and efficient production of DEH will ensure an optimal process and sufficient quantities of end products. We have previously cloned and characterized endo-type Alg7D [17] and exo-type Alg17C [18] alginate lyases in the marine bacterium Saccharophagus degradans 2-40T. In the present study, we have optimized the enzymatic saccharification process using recombinant Alg7D and Alg17C, thus leading to efficient production of DEH. This strategy of using two alginate lyases can be further combined with engineered microorganisms to metabolize DEH as a substrate for the synthesis of biofuels and chemicals.
Materials and methods Microbial strains and cultivation The recombinant bacterial strains Escherichia coli BL21(DE3) harboring alg7D and alg17C genes, which were previously constructed [17, 18], were used for the production of Alg7D and Alg17C enzymes, respectively. The cells were grown in Luria–Bertani broth (BD, Sparks, MD, USA) containing 50 mg/L ampicillin in a shaking
Bioprocess Biosyst Eng
Purification of recombinant proteins Cells were disrupted by ultrasonication and were centrifuged at 16,0009g for 1 h. The cell-free supernatant was passed through a HisTrap column (GE Healthcare, Piscataway, NJ, USA) following the manufacturer’s protocol. Purified recombinant protein was concentrated using an Amicon Ultra Centrifugal Filter Unit (UFC903024, MW cutoff of 30 kDa; Millipore, Billerica, MA, USA). The concentration of purified protein was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Activity measurement of Alg7D One unit (U) of Alg7D lyase was defined as the amount of enzyme required to release 1 lmol reducing sugar per minute at pH 7.0 and 50 °C [17]. Enzyme loadings of Alg7D were in the range of 0.29–1.74 U/mL on 2 % (w/v) sodium alginate as substrate in 20 mM Tris–HCl buffer (pH 7.0) at time points ranging from 10 to 300 min. Reactions were quenched by submerging the tubes in boiling water for 5 min. The activity of Alg7D was measured by detecting the reducing sugar released from alginate using the 3,5-dinitrosalicylic acid (DNS) assay. Glucose was used as a standard in the DNS assay. Activity measurement of Alg17C Alg17C activity was measured following the method described in our previous study [17, 18]. In brief, alginate oligosaccharides produced by the reaction with Alg7D lyase were used as substrates. For DEH production, the reaction mixture was adjusted to pH 6.0, Alg17C lyase was added, and incubation was carried out at the optimal temperature of 40 °C. Enzyme loadings of Alg17C were in the range of 0.46–6.36 U/mL. Reactions were quenched by submerging the reaction tubes in boiling water for 5 min. The activity of Alg17C was measured using the DNS assay by detecting the reducing sugar produced in the reaction. Thin layer chromatography of enzymatic reaction products Thin layer chromatography (TLC) was conducted on silica gel 60 plates (Merck, Darmstadt, Germany). After
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incubator at 37 °C and 200 rpm until the absorbance at 600 nm of the culture broth reached 0.6. The cells were then incubated with 1 mM IPTG at 16 °C and 180 rpm for 18 h to induce recombinant enzyme expression. Thereafter, cells were collected by centrifugation and stored at -20 °C for recovery of recombinant proteins.
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Enzyme loading (U/mL) Fig. 2 Effect of enzyme loading on the initial velocity of enzymatic saccharification of 2 % (w/v) sodium alginate using Alg7D at pH 7.0 and 50 °C measured at different enzyme loadings
the TLC run, the separated reaction products were developed by placing the plates in a chamber containing a solution of n-butanol–acetic acid–water (3:2:2, v/v/v) for 1 h, followed by drying of the plates. Reaction products were visualized with a sulfuric acid–ethanol (1:4, v/v) solution. Gas chromatography–mass spectrometry of enzymatic reaction products Gas chromatography–mass spectrometry (GC–MS) was used for the identification of DEH produced by Alg17C from alginate oligosaccharides. DEH (50–1,000 lg) was derivatized prior to GC–MS analysis. During derivatization, the aldehyde group of DEH was protected by methoxyamination with 50 lL of 2 % (w/v) of methoxyamine hydrochloride in pyridine (Sigma, St. Louis, MO, USA) at 75 °C for 30 min. The volatility of DEH was increased by adding 80 lL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (Fluka, St. Louis, MO, USA) to the sample. The analysis was performed using a GC–MS (Agilent 7890A GC/5975C MSD system; Agilent Technologies, Wilmington, DE, USA), equipped with a DB-5ms column (I.D. 30 m 9 0.25 mm, 0.25-lm film thickness; Agilent Technologies). The column temperature was programmed as follows: 100 °C for 3.5 min; ramped up to 160 °C at 15 °C/min and maintained for 20 min; ramped up to 200 °C at 20 °C/min and maintained for 15 min; and finally ramped up to 280 °C at 20 °C/min and maintained for 5 min. Acquisition of the mass spectra was in the range of 50–700 m/ z at 70 eV electron impact and ion source temperature of 230 °C.
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Bioprocess Biosyst Eng
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Results and discussion Optimization of Alg7D enzyme loading during oligosaccharide production
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Fig. 3 Effect of enzyme loading on the initial velocity of enzymatic saccharification of alginate oligosaccharides using Alg17C at pH 6.0 and 40 °C measured at different enzyme loadings
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Enzyme loading of Alg7D was investigated at the optimal reaction conditions for Alg7D (pH 7.0 and 50 °C), as determined previously [17], for efficient production of alginate oligomers. Figure 2 shows the initial velocities of formation of reducing sugar at different enzyme loadings of Alg7D using 2 % (w/v) of sodium alginate as a substrate. The initial velocity rose almost linearly until 1.16 U/ mL of Alg7D, beyond which it remained constant. Therefore, 1.16 U/mL of Alg7D was selected as the optimal loading value for further studies, in which the initial velocity and the theoretical maximum conversion to reducing sugar were 0.17 mg/mL-min and 20.8 %, respectively. The alginate oligosaccharides (mainly DP4 and DP5) from these initial conversion reactions served as substrates in subsequent reactions with the exo-type alginate lyase Alg17C for producing monosaccharides.
The conversion efficiency for 3.68 U/mL of Alg17C was 45.6 % of the theoretical maximum conversion to reducing sugar.
Optimization of Alg17C enzyme loading during monosaccharide production
Sequential or simultaneous addition of Alg7D and Alg17C for alginate saccharification
Alg17C is an exo-type alginate lyase that can produce alginate monomers from oligomers [18]. To optimize the process of conversion to monomers, different loading amounts of Alg17C were tested at pH 6.0 and 40 °C, using alginate oligosaccharides (from the reaction using Alg7D) as substrates. As shown in Fig. 3, the initial velocity rose to a maximum of 0.17 g/L-min at 3.68 U/mL of Alg17C. Therefore, the optimal Alg17C loading activity of 3.68 U/ mL was used for all subsequent saccharification reactions.
We evaluated whether sequential reactions or simultaneous addition of the two enzymes, Alg7D and Alg17C, yielded a more efficient conversion to DEH. Figure 4a shows the results from the sequential addition of Alg7D and Alg17C in the reaction mixture for the saccharification of alginate to DEH. Preliminary experiments showed significant loss of activity for Alg17C at 50 °C (data not shown), which is the optimal temperature for Alg7D; hence, all subsequent saccharification reactions were carried out at 40 °C and pH
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pH 6.5 and 45°C
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Reaction time (min) Fig. 4 Production of reducing sugar from 2 % (w/v) sodium alginate. a Alg7D and Alg17C were sequentially added for the saccharification process at pH 6.0 and 40 °C. b Alg7D and Alg17C were simultaneously added at different pHs and temperatures. For both (a) and (b), enzyme loadings of Alg7D and Alg17C were 1.16 and 3.68 U/mL, respectively
Bioprocess Biosyst Eng
Fig. 5 Display of reaction products converted from alginate oligosaccharides incubated for different time point with 3.68 U/mL of Alg17C by a TLC analysis and GC–MS analysis at b 1 min, c 10 min and d 30 min
6.0. Figure 4a shows results from the incubation of 1.16 U/ mL Alg7D with 2 % (w/v) sodium alginate until saturation of reducing sugars was reached at 60 min. This was followed by the addition of 3.68 U/mL of Alg17C, with no change in reaction conditions. Production of reducing sugar continued for 80 min and finally reached a maximum concentration of 8.09 g/L, which represents 45.5 % of the theoretical maximum conversion yield of DEH from alginate. Next, the simultaneous addition of Alg7D and Alg17C for the conversion to DEH was investigated, because it would shorten the saccharification time and simplify the process as compared to the sequential addition of the two enzymes. The reaction conditions for the simultaneous addition of Alg7D and Alg17C to the reaction mixture tested were (1) optimum for Alg7D, pH 7.0 and 50 °C; (2) optimum for Alg17C, pH 6.0 and 40 °C and (3) midpoints of the optima for Alg7D and Alg17C, pH 6.5 and 45 °C (Fig. 4b). The initial velocity differed depending on the pHs and temperatures used. The reaction conditions that were optimal for Alg7D (i.e., pH 7.0 and 50 °C) recorded the highest initial velocity. However, after 40 min, all the three reactions conditions reached saturation and produced similar amounts of reducing sugar, such as 5.06 g/L at pH 6.0, 40 °C; 5.49 g/L at pH 7.0, 50 °C and 5.12 g/L at pH 6.5, 45 °C, possibly due to the instability of Alg17C as mentioned earlier. The final reducing sugar concentrations corresponded to 28.5, 30.9 and 28.8 % of the theoretical
maximum yields of DEH from alginate (Fig. 4b). These yields from the simultaneous addition of the two enzymes were substantially lower than the conversion yield from the sequentially added enzymes (i.e., 45.5 % of theoretical maximum). In conclusion, sequential additions of Alg7D and Alg17C gave superior conversions, as compared to simultaneous additions, as the preferred method for obtaining higher yields of DEH. Analysis of saccharification reaction products by TLC and GC–MS TLC was used for analyzing the reaction products of sodium alginate from the sequential enzymatic reactions. The results of the analysis showed that alginate was depolymerized to oligosaccharides by Alg7D and then to monosaccharides by Alg17C (Fig. 5a). At 0 min, the main products were DP3, DP4, DP5, and other oligosaccharides with higher DPs. After incubation with Alg17C for 10 min, these oligosaccharides were converted to lower DP products and finally into monomers (presumably DEH). Disappearance of the oligosaccharides with DP3, DP4, and DP5 and appearance of the monomers correlated well with the addition and action of Alg17C. GC–MS analytical results also showed the abundance increase of the DEH-presumed peak as the enzymatic reaction progressed with time (Fig. 5b–d). For the structural verification of the monomeric product from the reaction of alginate saccharification,
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Bioprocess Biosyst Eng Fig. 6 Mass spectra of a 4deoxy-L-erythro-5-hexoseulose uronic acid (DEH) and b 2-keto3-deoxy-D-gluconic acid (KDG)
2-keto-3-deoxy-gluconic acid (KDG), a monomeric uronic acid, with a highly similar chemical structure to that of DEH, was used as a standard. It was because the mass spectra and authentic standard for DEH were not available. KDG showed a highly similar mass spectrum to that of the reaction product (Fig. 6); therefore, it was deduced that the mass spectrum of the monomer corresponded to DEH.
analysis showed that the reaction products were predominantly monomers, and GC–MS analysis revealed that the mass spectra of the monomers corresponded to DEH. DEH can be produced efficiently using recombinant enzymes, and fermentative microorganisms metabolically engineered to catabolize DEH could be further exploited for the mass production of third-generation biofuels using brown macroalgae.
Conclusion
Acknowledgments This work was supported by grants from the Ministry of Oceans and Fisheries (20131039449) and the Advanced Biomass R&D Center of Korea (2011-0031353) funded the Ministry of Science, ICT & Future Planning. Facility support by the Institute of Biomedical Science and Food Safety at Korea University Food Safety Hall is acknowledged.
In the current study, we established and optimized an enzymatic saccharification strategy for the production of DEH from alginate that is the main component of brown macroalgae. Optimization of enzyme loading was carried out by measuring the initial velocity of each reaction up to saturation of product formation. Sequential addition of the endo- and exo-alginate lyases (Alg7D and Alg17C, respectively) gave a better conversion yield than the simultaneous addition of the enzymes (45.5 % vs. 30.9 %, respectively, of the theoretical maximum yield). TLC
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References 1. Food and Agriculture Organization of United Nations (2009) How to Feed the World in 2050 2. Jung YH, Kim S, Yang TH, Lee HJ, Seung D, Park Y-C, Seo J-H, Choi I-G, Kim KH (2012) Aqueous ammonia pretreatment,
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saccharification, and fermentation evaluation of oil palm fronds for ethanol production. Bioprocess Biosyst Eng 35:1497–1503 Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D (2011) Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol 102:10163–10172 Ramnani P, Chitarrari R, Tuohy K, Grant J, Hotchkiss S, Philp K, Campbell R, Gill C, Rowland I (2012) In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar and alginate seaweeds. Anaerobe 18:1–6 Fukuda H, Imura M, Kasahara H, Kirimura K, Usami S (1987) Effects of the addition of laminaran on b-glucosidase production by Trichoderma viride. J Ferment Technol 65:91–93 Ghose TK, Roychoudhury PK, Ghosh P (1984) Simultaneous saccharification and fermentation (SSF) of lignocellulosics to ethanol under vacuum cycling and step feeding. Biotechnol Bioeng 26:377–381 Andriamanantoanina H, Rinaudo M (2010) Characterization of the alginates from five Madagascan brown algae. Carbohydr Polym 82:555–560 Gacesa P (1988) Alginates. Carbohydr Polym 8:161–182 Okuda K, Oka K, Onda A, Kajiyoshi K, Hiraoka M, Yanagisawa K (2008) Hydrothermal fractional pretreatment of sea algae and its enhanced enzymatic hydrolysis. J Chem Technol Biotechnol 83:836–841 Aida TM, Yamagata T, Watanabe M, Smith Jr RL (2010) Depolymerization of sodium alginate under hydrothermal conditions. Carbohydr Polym 80:296–302 Haug A, Larsen B, Smidsrod O (1966) A study of the constitution of alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190 Miyake O, Hashimoto W, Murata K (2003) An exotype alginate lyase in Sphingomonas sp. Al: overexpression in Escherichia
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coli, purification, and characterization of alginate lyase IV (A1IV). Protein Expr Purif 29:33–41 Kersters K, De Ley J (1968) The occurrence of the Entner– Doudoroff pathway in bacteria. Antonie Van Leeuwenhoek 34:393–408 Takeda H, Yoneyama F, Kawai S, Hashimoto W, Murata K (2011) Bioethanol production from marine biomass alginate by metabolically engineered bacteria. Energy Environ Sci 4:2575–2581 Wargacki AJ, Leonard E, Win MN, Regitsky DD, Santos CNS, Kim PB, Cooper SR, Raisner RM, Herman A, Sivitz AB, Lakshmanaswamy A, Kashiyama Y, Baker D, Yoshikuni Y (2012) An engineered microbial platform for direct biofuel production from brown macroalgae. Science 335:308–313 Enguist-Newman M, Faust AME, Bravo DD, Santos CNS, Raisner RM, Hanel A, Sarvabhowman P, Le C, Regitsky DD, Cooper SR, Peereboom L, Clark A, Martinez Y, Goldsmith J, Cho MY, Donohoue PD, Luo L, Lamberson B, Tamrakar P, Kim EJ, Villari JL, Gill A, Tripathi SA, Karamchedu P, Paredes CJ, Rajgarhia V, Kotlar HK, Bailey RB, Miller DJ, Ohler NL, Swimmer C, Yoshikuni Y (2014) Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505:239–243 Kim HT, Ko H-J, Kim N, Kim D, Lee D, Choi I-G, Woo HC, Kim MD, Kim KH (2012) Characterization of a recombinant endo-type alginate lyase (Alg7D) from Saccharophagus degradans. Biotechnol Lett 34:1087–1092 Kim HT, Chung JH, Wang D, Lee J, Woo HC, Choi I-G, Kim KH (2012) Depolymerization of alginate into a monomeric sugar acid using Alg17C, an exo-oligoalginate lyase cloned from Saccharophagus degradans 2-40. Appl Microbiol Biotechnol 93:2233–2239
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ORIGINAL PAPER
Optimal production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases Da Mao Wang • Hee Taek Kim • Eun Ju Yun Do Hyoung Kim • Yong-Cheol Park • Hee Chul Woo • Kyoung Heon Kim
•
Received: 9 October 2013 / Accepted: 6 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography–mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of
D. M. Wang E. J. Yun D. H. Kim K. H. Kim (&) Department of Biotechnology, Korea University Graduate School, Seoul 136-713, Republic of Korea e-mail: [email protected] H. T. Kim Research Center for Biobased Chemistry, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea Y.-C. Park Department of Bio and Fermentation Convergence, Kookmin University, Seoul 136-702, Republic of Korea H. C. Woo Department of Chemical Engineering, Pukyung National University, Busan 608-739, Republic of Korea
DEH that is the key substrate for producing biofuels from brown macro algal biomass. Keywords Alginate lyase Brown macro algae Uronic acid Saccharification Saccharophagus degradans
Introduction Biofuel production using first-generation biomass such as sugar and starch can lead to conflicts with food production due to the increasing worldwide food consumption [1]. Alternatively, second-generation biomass represented by lignocellulose is actively considered, but it requires substantial pretreatment steps [2]. Algal biomass has been gaining importance as a third-generation biofuel resource for the production of fuels and chemicals [3]. In particular, macroalgae, which contain a large amount of carbohydrates, can be utilized as sources of fermentable sugars [4–6]. The dominant carbohydrate in brown macroalgae is alginate, which comprises 12–34 % of its total dry mass [7]. Alginate is a linear polysaccharide composed of b-D-mannuronic acid (M) and its C5 epimer, a-L-guluronic acid (G) [8]. An important prerequisite step for alginate metabolism by microorganisms is its depolymerization into sugars with low degrees of polymerization (DP), such as oligosaccharides and monosaccharides. The most commonly employed processes for alginate depolymerization are thermochemical and biochemical conversions. Thermochemical conversions are single-step processes that consist of hydrothermal treatments [9, 10] with catalysts, such as oxalic acid [11], which result in the formation of oligomers as well as byproducts, such as lactic acid, glycolic acid, and other water-soluble substances.
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Bioprocess Biosyst Eng Fig. 1 Schematic diagram for the enzymatic depolymerization of alginate using endo- and exotype alginate lyases
In contrast, biochemical depolymerization of alginate is a multi-step process that includes enzymatic and nonenzymatic conversions. The enzymatic depolymerization process is carried out using alginate lyases (Fig. 1). These lyases can be classified into two major groups based on the mode of degradation of alginate: endo-type alginate lyases cleave within the chain and produce unsaturated uronic acid oligomers with a double bond between C4 and C5 at the nonreducing end, whereas exo-type alginate lyases cleave at the termini of the chains to produce monosaccharides from oligosaccharides. The monomers are then nonenzymatically converted into an a-ketouronate, 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) [12]. DEH is a key starting material, which can be converted into 2-keto-3-deoxy-D-gluconate (KDG) by a reductase. KDG, another important intermediate, enters the Entner–Doudoroff pathway to generate building blocks and energy for cellular metabolism [13]. DEH is the most abundant substrate obtainable from brown macroalgae, and this sugar acid that is not fermented by general microorganisms is now becoming convertible into ethanol or other chemicals using recombinant microorganisms [14–16]. Therefore, DEH is the key player in the fuel production using brown macro algal
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biomass, and efficient production of DEH will ensure an optimal process and sufficient quantities of end products. We have previously cloned and characterized endo-type Alg7D [17] and exo-type Alg17C [18] alginate lyases in the marine bacterium Saccharophagus degradans 2-40T. In the present study, we have optimized the enzymatic saccharification process using recombinant Alg7D and Alg17C, thus leading to efficient production of DEH. This strategy of using two alginate lyases can be further combined with engineered microorganisms to metabolize DEH as a substrate for the synthesis of biofuels and chemicals.
Materials and methods Microbial strains and cultivation The recombinant bacterial strains Escherichia coli BL21(DE3) harboring alg7D and alg17C genes, which were previously constructed [17, 18], were used for the production of Alg7D and Alg17C enzymes, respectively. The cells were grown in Luria–Bertani broth (BD, Sparks, MD, USA) containing 50 mg/L ampicillin in a shaking
Bioprocess Biosyst Eng
Purification of recombinant proteins Cells were disrupted by ultrasonication and were centrifuged at 16,0009g for 1 h. The cell-free supernatant was passed through a HisTrap column (GE Healthcare, Piscataway, NJ, USA) following the manufacturer’s protocol. Purified recombinant protein was concentrated using an Amicon Ultra Centrifugal Filter Unit (UFC903024, MW cutoff of 30 kDa; Millipore, Billerica, MA, USA). The concentration of purified protein was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Activity measurement of Alg7D One unit (U) of Alg7D lyase was defined as the amount of enzyme required to release 1 lmol reducing sugar per minute at pH 7.0 and 50 °C [17]. Enzyme loadings of Alg7D were in the range of 0.29–1.74 U/mL on 2 % (w/v) sodium alginate as substrate in 20 mM Tris–HCl buffer (pH 7.0) at time points ranging from 10 to 300 min. Reactions were quenched by submerging the tubes in boiling water for 5 min. The activity of Alg7D was measured by detecting the reducing sugar released from alginate using the 3,5-dinitrosalicylic acid (DNS) assay. Glucose was used as a standard in the DNS assay. Activity measurement of Alg17C Alg17C activity was measured following the method described in our previous study [17, 18]. In brief, alginate oligosaccharides produced by the reaction with Alg7D lyase were used as substrates. For DEH production, the reaction mixture was adjusted to pH 6.0, Alg17C lyase was added, and incubation was carried out at the optimal temperature of 40 °C. Enzyme loadings of Alg17C were in the range of 0.46–6.36 U/mL. Reactions were quenched by submerging the reaction tubes in boiling water for 5 min. The activity of Alg17C was measured using the DNS assay by detecting the reducing sugar produced in the reaction. Thin layer chromatography of enzymatic reaction products Thin layer chromatography (TLC) was conducted on silica gel 60 plates (Merck, Darmstadt, Germany). After
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incubator at 37 °C and 200 rpm until the absorbance at 600 nm of the culture broth reached 0.6. The cells were then incubated with 1 mM IPTG at 16 °C and 180 rpm for 18 h to induce recombinant enzyme expression. Thereafter, cells were collected by centrifugation and stored at -20 °C for recovery of recombinant proteins.
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Enzyme loading (U/mL) Fig. 2 Effect of enzyme loading on the initial velocity of enzymatic saccharification of 2 % (w/v) sodium alginate using Alg7D at pH 7.0 and 50 °C measured at different enzyme loadings
the TLC run, the separated reaction products were developed by placing the plates in a chamber containing a solution of n-butanol–acetic acid–water (3:2:2, v/v/v) for 1 h, followed by drying of the plates. Reaction products were visualized with a sulfuric acid–ethanol (1:4, v/v) solution. Gas chromatography–mass spectrometry of enzymatic reaction products Gas chromatography–mass spectrometry (GC–MS) was used for the identification of DEH produced by Alg17C from alginate oligosaccharides. DEH (50–1,000 lg) was derivatized prior to GC–MS analysis. During derivatization, the aldehyde group of DEH was protected by methoxyamination with 50 lL of 2 % (w/v) of methoxyamine hydrochloride in pyridine (Sigma, St. Louis, MO, USA) at 75 °C for 30 min. The volatility of DEH was increased by adding 80 lL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (Fluka, St. Louis, MO, USA) to the sample. The analysis was performed using a GC–MS (Agilent 7890A GC/5975C MSD system; Agilent Technologies, Wilmington, DE, USA), equipped with a DB-5ms column (I.D. 30 m 9 0.25 mm, 0.25-lm film thickness; Agilent Technologies). The column temperature was programmed as follows: 100 °C for 3.5 min; ramped up to 160 °C at 15 °C/min and maintained for 20 min; ramped up to 200 °C at 20 °C/min and maintained for 15 min; and finally ramped up to 280 °C at 20 °C/min and maintained for 5 min. Acquisition of the mass spectra was in the range of 50–700 m/ z at 70 eV electron impact and ion source temperature of 230 °C.
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Results and discussion Optimization of Alg7D enzyme loading during oligosaccharide production
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Fig. 3 Effect of enzyme loading on the initial velocity of enzymatic saccharification of alginate oligosaccharides using Alg17C at pH 6.0 and 40 °C measured at different enzyme loadings
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Enzyme loading of Alg7D was investigated at the optimal reaction conditions for Alg7D (pH 7.0 and 50 °C), as determined previously [17], for efficient production of alginate oligomers. Figure 2 shows the initial velocities of formation of reducing sugar at different enzyme loadings of Alg7D using 2 % (w/v) of sodium alginate as a substrate. The initial velocity rose almost linearly until 1.16 U/ mL of Alg7D, beyond which it remained constant. Therefore, 1.16 U/mL of Alg7D was selected as the optimal loading value for further studies, in which the initial velocity and the theoretical maximum conversion to reducing sugar were 0.17 mg/mL-min and 20.8 %, respectively. The alginate oligosaccharides (mainly DP4 and DP5) from these initial conversion reactions served as substrates in subsequent reactions with the exo-type alginate lyase Alg17C for producing monosaccharides.
The conversion efficiency for 3.68 U/mL of Alg17C was 45.6 % of the theoretical maximum conversion to reducing sugar.
Optimization of Alg17C enzyme loading during monosaccharide production
Sequential or simultaneous addition of Alg7D and Alg17C for alginate saccharification
Alg17C is an exo-type alginate lyase that can produce alginate monomers from oligomers [18]. To optimize the process of conversion to monomers, different loading amounts of Alg17C were tested at pH 6.0 and 40 °C, using alginate oligosaccharides (from the reaction using Alg7D) as substrates. As shown in Fig. 3, the initial velocity rose to a maximum of 0.17 g/L-min at 3.68 U/mL of Alg17C. Therefore, the optimal Alg17C loading activity of 3.68 U/ mL was used for all subsequent saccharification reactions.
We evaluated whether sequential reactions or simultaneous addition of the two enzymes, Alg7D and Alg17C, yielded a more efficient conversion to DEH. Figure 4a shows the results from the sequential addition of Alg7D and Alg17C in the reaction mixture for the saccharification of alginate to DEH. Preliminary experiments showed significant loss of activity for Alg17C at 50 °C (data not shown), which is the optimal temperature for Alg7D; hence, all subsequent saccharification reactions were carried out at 40 °C and pH
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pH 6.5 and 45°C
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Reaction time (min) Fig. 4 Production of reducing sugar from 2 % (w/v) sodium alginate. a Alg7D and Alg17C were sequentially added for the saccharification process at pH 6.0 and 40 °C. b Alg7D and Alg17C were simultaneously added at different pHs and temperatures. For both (a) and (b), enzyme loadings of Alg7D and Alg17C were 1.16 and 3.68 U/mL, respectively
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Fig. 5 Display of reaction products converted from alginate oligosaccharides incubated for different time point with 3.68 U/mL of Alg17C by a TLC analysis and GC–MS analysis at b 1 min, c 10 min and d 30 min
6.0. Figure 4a shows results from the incubation of 1.16 U/ mL Alg7D with 2 % (w/v) sodium alginate until saturation of reducing sugars was reached at 60 min. This was followed by the addition of 3.68 U/mL of Alg17C, with no change in reaction conditions. Production of reducing sugar continued for 80 min and finally reached a maximum concentration of 8.09 g/L, which represents 45.5 % of the theoretical maximum conversion yield of DEH from alginate. Next, the simultaneous addition of Alg7D and Alg17C for the conversion to DEH was investigated, because it would shorten the saccharification time and simplify the process as compared to the sequential addition of the two enzymes. The reaction conditions for the simultaneous addition of Alg7D and Alg17C to the reaction mixture tested were (1) optimum for Alg7D, pH 7.0 and 50 °C; (2) optimum for Alg17C, pH 6.0 and 40 °C and (3) midpoints of the optima for Alg7D and Alg17C, pH 6.5 and 45 °C (Fig. 4b). The initial velocity differed depending on the pHs and temperatures used. The reaction conditions that were optimal for Alg7D (i.e., pH 7.0 and 50 °C) recorded the highest initial velocity. However, after 40 min, all the three reactions conditions reached saturation and produced similar amounts of reducing sugar, such as 5.06 g/L at pH 6.0, 40 °C; 5.49 g/L at pH 7.0, 50 °C and 5.12 g/L at pH 6.5, 45 °C, possibly due to the instability of Alg17C as mentioned earlier. The final reducing sugar concentrations corresponded to 28.5, 30.9 and 28.8 % of the theoretical
maximum yields of DEH from alginate (Fig. 4b). These yields from the simultaneous addition of the two enzymes were substantially lower than the conversion yield from the sequentially added enzymes (i.e., 45.5 % of theoretical maximum). In conclusion, sequential additions of Alg7D and Alg17C gave superior conversions, as compared to simultaneous additions, as the preferred method for obtaining higher yields of DEH. Analysis of saccharification reaction products by TLC and GC–MS TLC was used for analyzing the reaction products of sodium alginate from the sequential enzymatic reactions. The results of the analysis showed that alginate was depolymerized to oligosaccharides by Alg7D and then to monosaccharides by Alg17C (Fig. 5a). At 0 min, the main products were DP3, DP4, DP5, and other oligosaccharides with higher DPs. After incubation with Alg17C for 10 min, these oligosaccharides were converted to lower DP products and finally into monomers (presumably DEH). Disappearance of the oligosaccharides with DP3, DP4, and DP5 and appearance of the monomers correlated well with the addition and action of Alg17C. GC–MS analytical results also showed the abundance increase of the DEH-presumed peak as the enzymatic reaction progressed with time (Fig. 5b–d). For the structural verification of the monomeric product from the reaction of alginate saccharification,
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Bioprocess Biosyst Eng Fig. 6 Mass spectra of a 4deoxy-L-erythro-5-hexoseulose uronic acid (DEH) and b 2-keto3-deoxy-D-gluconic acid (KDG)
2-keto-3-deoxy-gluconic acid (KDG), a monomeric uronic acid, with a highly similar chemical structure to that of DEH, was used as a standard. It was because the mass spectra and authentic standard for DEH were not available. KDG showed a highly similar mass spectrum to that of the reaction product (Fig. 6); therefore, it was deduced that the mass spectrum of the monomer corresponded to DEH.
analysis showed that the reaction products were predominantly monomers, and GC–MS analysis revealed that the mass spectra of the monomers corresponded to DEH. DEH can be produced efficiently using recombinant enzymes, and fermentative microorganisms metabolically engineered to catabolize DEH could be further exploited for the mass production of third-generation biofuels using brown macroalgae.
Conclusion
Acknowledgments This work was supported by grants from the Ministry of Oceans and Fisheries (20131039449) and the Advanced Biomass R&D Center of Korea (2011-0031353) funded the Ministry of Science, ICT & Future Planning. Facility support by the Institute of Biomedical Science and Food Safety at Korea University Food Safety Hall is acknowledged.
In the current study, we established and optimized an enzymatic saccharification strategy for the production of DEH from alginate that is the main component of brown macroalgae. Optimization of enzyme loading was carried out by measuring the initial velocity of each reaction up to saturation of product formation. Sequential addition of the endo- and exo-alginate lyases (Alg7D and Alg17C, respectively) gave a better conversion yield than the simultaneous addition of the enzymes (45.5 % vs. 30.9 %, respectively, of the theoretical maximum yield). TLC
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