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Garabed Antranikian - One of the best experts on this subject based on the ideXlab platform.
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cloning sequencing and characterization of a heat and alkali stable type i Pullulanase from anaerobranca gottschalkii
Applied and Environmental Microbiology, 2004Co-Authors: Costanzo Bertoldo, Garabed Antranikian, Martin Armbrecht, Fiona Becker, Thomas Schafer, Wolfgang LieblAbstract:The gene encoding a type I Pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the Pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The Pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant Pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the Pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I Pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The Pullulanase from A. gottschalkii is the first thermoalkalistable type I Pullulanase that has been described.
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starch hydrolyzing enzymes from thermophilic archaea and bacteria
Current Opinion in Chemical Biology, 2002Co-Authors: Costanzo Bertoldo, Garabed AntranikianAbstract:Abstract Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including α-amylase, α-glucosidase, glucoamylase, Pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal Pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.
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A New Thermoactive Pullulanase from Desulfurococcus mucosus: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis
Journal of bacteriology, 2000Co-Authors: Fiona Duffner, Costanzo Bertoldo, Jens Tonne Andersen, Karen Wagner, Garabed AntranikianAbstract:The gene encoding a thermoactive Pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the Pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amyloPullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive Pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal Pullulanase type II with a wider substrate specificity.
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Pullulanase type i from fervidobacterium pennavorans ven5 cloning sequencing and expression of the gene and biochemical characterization of the recombinant enzyme
Applied and Environmental Microbiology, 1999Co-Authors: Costanzo Bertoldo, Fiona Duffner, Per Lina Jorgensen, Garabed AntranikianAbstract:The gene encoding the type I Pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The Pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with Pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified Pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, Pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
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heat stable Pullulanase from bacillus acidopullulyticus characterization and refolding after guanidinium chloride induced unfolding
Extremophiles, 1999Co-Authors: M Stefanova, Garabed Antranikian, Ruth M Schwerdtfeger, Roberto ScandurraAbstract:Heat-stable Pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60 degrees C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na2SO4 strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus Pullulanase was very sensitive to GdmC1 denaturation and had a transition midpoint at 1.2M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture: 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded Pullulanase.
Costanzo Bertoldo - One of the best experts on this subject based on the ideXlab platform.
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cloning sequencing and characterization of a heat and alkali stable type i Pullulanase from anaerobranca gottschalkii
Applied and Environmental Microbiology, 2004Co-Authors: Costanzo Bertoldo, Garabed Antranikian, Martin Armbrecht, Fiona Becker, Thomas Schafer, Wolfgang LieblAbstract:The gene encoding a type I Pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the Pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The Pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant Pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the Pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I Pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The Pullulanase from A. gottschalkii is the first thermoalkalistable type I Pullulanase that has been described.
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starch hydrolyzing enzymes from thermophilic archaea and bacteria
Current Opinion in Chemical Biology, 2002Co-Authors: Costanzo Bertoldo, Garabed AntranikianAbstract:Abstract Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including α-amylase, α-glucosidase, glucoamylase, Pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal Pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.
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A New Thermoactive Pullulanase from Desulfurococcus mucosus: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis
Journal of bacteriology, 2000Co-Authors: Fiona Duffner, Costanzo Bertoldo, Jens Tonne Andersen, Karen Wagner, Garabed AntranikianAbstract:The gene encoding a thermoactive Pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the Pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amyloPullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive Pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal Pullulanase type II with a wider substrate specificity.
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Pullulanase type i from fervidobacterium pennavorans ven5 cloning sequencing and expression of the gene and biochemical characterization of the recombinant enzyme
Applied and Environmental Microbiology, 1999Co-Authors: Costanzo Bertoldo, Fiona Duffner, Per Lina Jorgensen, Garabed AntranikianAbstract:The gene encoding the type I Pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The Pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with Pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified Pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, Pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
Zhengyu Jin - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of tryptophan437 at subsite 2 in Pullulanase from bacillus subtilis str 168
International Journal of Biological Macromolecules, 2019Co-Authors: Yuxiang Bai, Jinpeng Wang, Yinglu Cui, Zhengyu JinAbstract:Abstract Pullulanase, a typical debranching enzyme, specifically hydrolyzes α-1,6 glycosidic linkages in pullulan, starch and so on. There is accumulated knowledge about the catalysis of Pullulanase, but functional roles of acceptor subsites get less attention. According to the crystal structure of Pullulanase from Bacillus subtilis str. 168, stacking interaction between tryptophan437 and glycosyl unit of α-CD was found. Trp437 located in conserved region III was highly conserved in Pullulanases. But functional role of this residue remains unclear. Site-directed mutagenesis was used to determine the function of Trp437. Replacement of Trp437 with glycine, phenylalanine, proline, arginine resulted in mutants with undetectable hydrolysis activity and reverse hydrolysis activity. The secondary structure of mutated proteins showed no difference from the wild-type Pullulanase, but they lost the capability to bind β-CD based on the ITC measurement. Molecular docking was performed to investigate the affinity of proteins to ligands including maltotriose, 62-α-D-maltotriosyl-maltotriose and α-CD, showing the affinity of mutants to ligands became weaker compared to that of PulA. And the hydrogen bond between Trp437 and Glu525 in PulA only was found in mutant W437R. These results show that Trp437 at subsite +2 plays crucial role in catalysis of PulA, and it has little tolerance to change.
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effect of chitosan molecular weight on the formation of chitosan Pullulanase soluble complexes and their application in the immobilization of Pullulanase onto fe3o4 κ carrageenan nanoparticles
Food Chemistry, 2016Co-Authors: Jie Long, Zhengyu Jin, Fang Wang, Aiquan Jiao, Xiaobei ZhanAbstract:The interactions between Pullulanase and chitosans of different molecular weights (Mw) were comprehensively studied, and their applications in Pullulanase immobilization onto Fe3O4-κ-carrageenan nanoparticles upon chitosan-Pullulanase complexation were also evaluated. Chitosan (CS) complexation with Pullulanase was found to be dependent on pH and chitosan Mw. The critical pH of structure-forming events during complexation shifted significantly (p<0.05) to a lower pH with a low Mw chitosan (50kDa) compared to other chitosan types. Binding constants for the chitosan-Pullulanase interaction increased in the following order: CS-500
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new method for the immobilization of Pullulanase onto hybrid magnetic fe3o4 κ carrageenan nanoparticles by electrostatic coupling with Pullulanase chitosan complex
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Jie Long, Zhengyu Jin, Aiquan JiaoAbstract:We present a simple method to immobilize Pullulanase onto hybrid magnetic (Fe3O4–κ-carrageenan) nanoparticles, involving the in situ synthesis of magnetic carrageenan nanoparticles and the formation of Pullulanase/chitosan complex. The complex behavior of Pullulanase with chitosan as a function of pH and protein–polysaccharide ratio was studied by turbidimetric titration. Then, the as-prepared immobilized enzymes were characterized by vibrating-sample magnetometer, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffractometer, and thermogravimetric analysis. It was found that the activity retention of immobilized Pullulanase and amount of enzyme loaded reached 95.5% and 96.3 mg/g, respectively, under optimal conditions. The immobilized enzyme exhibited great operational stability (retaining approximately 61% residual activity after ten consecutive reuses), demonstrating that enzyme leakage during the catalysis reaction was efficiently reduced. Furthermore, the activity of...
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preparation and identification of 62 α maltotriosyl maltotriose using a commercial Pullulanase
International Journal of Food Properties, 2015Co-Authors: Yuxiang Bai, Zhengyu JinAbstract:In this article, 62−α-maltotriosyl-maltotriose was consecutively prepared by hydrolysis and reverse synthesis action of Bacillus Acidopullulyticus Pullulanase using pullulan as a substrate. Maltotriose syrup was obtained by hydrolyzing of pullulan with Pullulanase. The optimal reverse synthesis conditions were then investigated and the optimum conditions were as follows: maltotriose syrup concentration, 70–75%; time, 40 h; pH, 4.0; temperature, 65°C; amount of Pullulanase, 120 U. Under these conditions, the relative conversion rate of reverse synthesis reached up to 26%. The products were further isolated by preparative high performance liquid chromatography on Lichrospher NH2 column. 62−α-Maltotriosyl-maltotriose was purified and maltotriose syrup was collected and recycled as the substrate for reverse synthesis. Structure of 62−α-maltotriosyl-maltotriose was elucidated by electrospray ionization mass spectrometry, fourier transform infrared spectroscopy, 1H nuclear magnetic resonance, 13C nuclear magnet...
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a novel method for Pullulanase immobilized onto magnetic chitosan fe3o4 composite nanoparticles by in situ preparation and evaluation of the enzyme stability
Journal of Molecular Catalysis B-enzymatic, 2014Co-Authors: Jie Long, Aiquan Jiao, Benxi Wei, Yujing Zhang, Zhengyu JinAbstract:Abstract A novel immobilization of Pullulanase has been developed by using magnetic chitosan/Fe 3 O 4 nanoparticles (CS-MNPs). The CS-MNPs were synthesized by in situ mineralization of Fe 3 O 4 in chitosan hydrogel and characterized by transmission electron microscopy, vibrating sample magnetometer, Fourier transform infrared spectroscopy, thermogravimetric analyzer and X-ray diffractometer. The resultant material (CS-MNPs) was used as support for the covalent immobilization of Pullulanase with glutaraldehyde as cross-linker. In the process of Pullulanase immobilization, both the amount (g) of immobilized Pullulanase and the activity retention(%) increased with enzyme concentration increasing (15–60 μg/ml), and the optimum activity retention (93.5%) was obtained at pH 3.5. In enzymatic properties, the immobilized enzyme exhibited a broadened pH profiles and higher thermal stability than the free enzyme. The optimum pH of immobilized Pullulanase (3.5) was lower than that of the free enzyme (4.4), and the immobilized enzymes could retain about 84% of their initial activity after incubated for 5 h at 60 °C in contrast with 58% of free enzyme. Furthermore, the immobilized Pullulanase retained more than 56% of its initial activity after eight consecutive reuses. These results prove that the CS-MNPs are an effective support for the immobilization of Pullulanase.
Xuguo Duan - One of the best experts on this subject based on the ideXlab platform.
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efficient extracellular expression of bacillus deramificans Pullulanase in brevibacillus choshinensis
Journal of Industrial Microbiology & Biotechnology, 2016Co-Authors: Chun Zou, Xuguo DuanAbstract:In this study, the Pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The Pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the Pullulanase activity and the specific Pullulanase productivity reached 1005.8 U/mL and 110.5 × 10(3) U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of Pullulanase.
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triton x 100 enhances the solubility and secretion ratio of aggregation prone Pullulanase produced in escherichia coli
Bioresource Technology, 2015Co-Authors: Xuguo Duan, Chun ZouAbstract:The Pullulanase from Bacillus deramificans is an industrially useful starch-debranching enzyme that is difficult to produce in large quantities. In this study, B. deramificans Pullulanase was found to be an aggregation-prone protein that can be solubilized from the insoluble fraction by surfactants in vitro. Studying the effects of various surfactants on Pullulanase production in Escherichia coli in shake flasks revealed that optimal Pullulanase production could be obtained by adding 0.5% Triton X-100 during the later period of fermentation. A modified fed-batch fermentation strategy was then applied to the production of Pullulanase in a 3-L fermentor. When supplemented with 0.5% Triton X-100 at 40 h, the maximal extracellular Pullulanase production and secretion ratio were 812.4 U mL(-1) and 86.0%, which were 46.2- and 47.8-fold that of the control, respectively.
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enhanced extracellular production of recombinant bacillus deramificans Pullulanase in escherichia coli through induction mode optimization and a glycine feeding strategy
Bioresource Technology, 2014Co-Authors: Xuguo Duan, Jing WuAbstract:Process optimization strategies were developed to improve extracellular production of recombinant Bacillus deramificans Pullulanase in Escherichia coli. Cell growth and Pullulanase production in shake-flask cultures were investigated as a function of the concentration of added glycine, and the type and concentration of inducer. From the results of these experiments, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 3-L fermentor. The gradual addition of lactose was utilized for the induction of protein expression. The optimal lactose feeding rate and induction point were 0.4gL(-1)h(-1) and a dry cell weight (DCW) of 15gL(-1), respectively. Furthermore, a glycine feeding strategy was formulated to promote the secretion of recombinant protein. The optimal total and extracellular Pullulanase activity were 2523.5 and 1567.9UmL(-1), respectively, which represent 1.2 and 22.6-fold increases compared with those observed under unoptimized conditions.
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optimization of Pullulanase production in escherichia coli by regulation of process conditions and supplement with natural osmolytes
Bioresource Technology, 2013Co-Authors: Xuguo Duan, Jian ChenAbstract:Abstract In this study, the effects of temperature, IPTG (Isopropyl β- d -1-thiogalactopyranoside) concentration, and osmolytes (proline, K-glutamate, and betaine) on cell growth and soluble Pullulanase productivity of recombinant Escherichia coli were investigated. The yield of soluble Pullulanase was found to be enhanced with decrease in cultivation temperature, lower IPTG concentration, and betaine supplementation in a shake flask. In addition, a modified two-stage feeding strategy was proposed and applied in a 3-L fermentor supplied with 20 mM betaine, which achieved a dry cell weight of 59.3 g L−1. Through this cultivation approach at 25 °C, the total soluble activity of Pullulanase reached 963.9 U mL−1, which was 8.3-fold higher than that observed without addition of betaine at 30 °C (115.8 U mL−1). The higher expression of soluble Pullulanase in a scalable semisynthetic medium showed the potential of the proposed process for the industrial production of soluble enzyme.
Yao Nie - One of the best experts on this subject based on the ideXlab platform.
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evolutionary coupling saturation mutagenesis coevolution guided identification of distant sites influencing bacillus naganoensis Pullulanase activity
FEBS Letters, 2020Co-Authors: Xinye Wang, Xiaoran Jing, Yi Deng, Yao Nie, Yilei Zhao, John F Hunt, Gaetano T Montelione, Thomas SzyperskiAbstract:Pullulanases are well-known debranching enzymes hydrolyzing α-1,6-glycosidic linkages. To date, engineering of Pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis Pullulanase as a target protein, here we introduce the 'evolutionary coupling saturation mutagenesis' (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.
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Improvement of the Activity and Stability of Starch-Debranching Pullulanase from Bacillus naganoensis via Tailoring of the Active Sites Lining the Catalytic Pocket
2018Co-Authors: Xinye Wang, Yao NieAbstract:Pullulanases are well-known debranching enzymes that hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. However, most of the Pullulanases exhibit limited activity for practical applications. Here, two sites (787 and 621) lining the catalytic pocket of Bacillus naganoensis Pullulanase were identified as being critical for enzymatic activity by triple-code saturation mutagenesis. Subsequently, both sites were subjected to NNK-based saturation mutagenesis to obtain positive variants. Among the variants showing enhanced activity, the enzymatic activity and specific activity of D787C were 1.5-fold higher than those of the wild-type (WT). D787C also showed a 1.8-fold increase in kcat and a 1.7-fold increase in kcat/Km. In addition, D787C maintained higher activity compared with that of WT at temperatures over 60 °C. All the positive variants showed higher acid resistance, with D787C maintaining 90% residual activity at pH 4.0. Thus, enzymes with improved properties were obtained by saturation mutagenesis at the active site
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enhancement of extracellular expression of bacillus naganoensis Pullulanase from recombinant bacillus subtilis effects of promoter and host
Protein Expression and Purification, 2016Co-Authors: Wan Song, Yao NieAbstract:Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of Pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis Pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed Pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on Pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular Pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on Pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis.
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single amino acid substitution in the Pullulanase of klebsiella variicola for enhancing thermostability and catalytic efficiency
Applied Biochemistry and Biotechnology, 2015Co-Authors: Yao Nie, Rong XiaoAbstract:Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 Pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type Pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the Pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of V max and k cat/K m, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the Pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently.
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enhancement of extracellular Pullulanase production from recombinant escherichia coli by combined strategy involving auto induction and temperature control
Bioprocess and Biosystems Engineering, 2014Co-Authors: Wenbo Chen, Yao Nie, Rong XiaoAbstract:Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-d-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml−1) and cell biomass (7.95 g DCW l−1). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular Pullulanase. Finally, with a view to releasing more intracellular Pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml−1, nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.
