A Novel Neutral and Mesophilic β-Glucosidase from Coral Microorganisms for Efficient Preparation of Gentiooligosaccharides

β-glucosidases can produce gentiooligosaccharides that are lucrative and promising for the prebiotic and alternative food industries. However, the commercial production of gentiooligosaccharides using β-glucosidase is challenging, as this process is limited by the need for high thermal energy and increasing demand for the enzyme. Here, a putative β-glucosidase gene, selected from the coral microbial metagenome, was expressed in Escherichia coli. Reverse hydrolysis of glucose by Blg163 at pH 7.0 and 40 °C achieved a gentiooligosaccharide yield of 43.02 ± 3.20 g·L−1 at a conversion rate of 5.38 ± 0.40%. Transglycosylation of mixed substrates, glucose and cellobiose, by Blg163 consumed 21.6 U/0.5 g glucose/g cellobiose, achieving a gentiooligosaccharide yield of 70.34 ± 2.20 g·L−1 at a conversion rate of 15.63%, which is close to the highest yield reported in previous findings. Blg163-mediated synthesis of gentiooligosaccharides is the mildest reaction and the lowest β-glucosidase consumption reported to date.

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10−3 mM s−1), and Kcat/Km (763.92 mM−1 s−1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wildtype. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4−(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity towards natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-acetyl COSs. Results: The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12−35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4,878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min-1 mg-1, 0.50 μmol mL-1, 25,555.56 s-1, and 51,111.12 mL μmol-1 s-1, respectively. Analysis of the hydrolysis products of N-acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-acetyl COSs ((GlcNAc)3−(GlcNAc)7) from (GlcNAc)2−(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions: The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggests a possible application in the production of GlcNAc or higher N-acetyl COSs.

A novel bacterial β- N -acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Background: N -acetyl glucosamine (GlcNAc) and N -acetyl chitooligosaccharides ( N -acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N -acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc) 4 −(GlcNAc) 7 ) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity towards natural killer cells. Thus, it is of great significance to discover a β- N -acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize higher N -acetyl COSs. Results: The gene encoding the novel β- N -acetyl glucosaminidase, designated C m NAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of C m NAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity with the corresponding domain of the well-characterized NAGases. The C m NAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified C m NAGase toward p -nitrophenyl- N -acetyl glucosaminide ( p NP-GlcNAc) was 4,878.6 U/mg of protein. C m NAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The V max , K m , K cat , and K cat / K m of C m NAGase for p NP-GlcNAc were 16,666.67 μmol min -1 mg -1 , 0.50 μmol mL -1 , 25,555.56 s -1 , and 51,111.12 mL μmol -1 s -1 , respectively. Analysis of the hydrolysis products of N -acetyl COSs and colloidal chitin revealed that C m NAGase is a typical exo-acting NAGase. Particularly, C m NAGase can synthesize higher N -acetyl COSs ((GlcNAc) 3 −(GlcNAc) 7 ) from (GlcNAc) 2 −(GlcNAc) 6 , respectively, showed that it possesses transglycosylation activity. In addition, C m NAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusion s : The observations recorded in this study that C m NAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggests a possible application in the production of GlcNAc or higher N -acetyl COSs.

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/ml/mg enzyme) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

<p>Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by <i>β</i>-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants<i> Tr</i>Cel1b^I177S, <i>Tr</i>Cel1b^I177S/I174S, and <i>Tr</i>Cel1b^{I177S/I174S/W173H}, and one negative variant <i>Tr</i>Cel1b^N240I were designed according to the <u>H</u>ydropathy <u>I</u>ndex <u>F</u>or <u>E</u>nzyme <u>A</u>ctivity (HIFEA) strategy. The reverse hydrolysis with <i>Tr</i>Cel1b^{I177S/I174S/W173H}was accelerated and then the maximum total production (<a>195.8 mg/ml/mg enzyme</a>) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, <a><i>Tr</i>Cel1b</a>^N240I lost reverse hydrolysis activity. The results demonstrate that<a> </a><a>the average hydropathy index</a> of <a>the key amino acid residues </a>in the catalytic site of<i> Tr</i>Cel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of <i>β</i>-glucosidases used for the synthesis of oligosaccharides.</p>

Efficient Synthesis of Rare Disaccharides by Engineered β-Glucosidase Based on Hydropathy Index

<p>Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis, especially rare disaccharides, is reverse hydrolysis by <i>β</i>-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants<i> Tr</i>Cel1b^I177S, <i>Tr</i>Cel1b^I177S/I174S, and <i>Tr</i>Cel1b^{I177S/I174S/W173H}, and one negative variant <i>Tr</i>Cel1b^N240I were designed according to the <u>H</u>ydropathy <u>I</u>ndex <u>F</u>or <u>E</u>nzyme <u>A</u>ctivity (HIFEA) strategy. The reverse hydrolysis with <i>Tr</i>Cel1b^{I177S/I174S/W173H}was accelerated and then the maximum total production (<a>195.8 mg/ml/mg enzyme</a>) of the synthesized disaccharides was increased 3.5-fold compared to that of wildtype. On the contrary, <a><i>Tr</i>Cel1b</a>^N240I lost reverse hydrolysis activity. The results demonstrate that<a> </a><a>the average hydropathy index</a> of <a>the key amino acid residues </a>in the catalytic site of<i> Tr</i>Cel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of <i>β</i>-glucosidases used for the synthesis of oligosaccharides.</p>