scholarly journals A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

2020 ◽  
Author(s):  
Alei Zhang ◽  
Xiaofang Mo ◽  
Ning Zhou ◽  
Yingying Wang ◽  
Guoguang Wei ◽  
...  
Keyword(s):  
Specific Activity ◽  
Antimicrobial Activities ◽  
Soluble Form ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Gene Encoding ◽  
Transglycosylation Activity ◽  
Catalytic Module ◽  
Hydrolase Family ◽  
Long Chain

Abstract Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit antitumor and antimicrobial activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Thus, it is crucial to discover a NAGase that can both synthesize GlcNAc and 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 with the corresponding domain of the well-characterized NAGases. CmNAGase gene was highly expressed with soluble form in Escherichia coli BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward p-nitrophenyl-N-acetyl glucosaminide. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, and Kcat of CmNAGase were 833.33 μmol·L-1 ·min-1, 10.9 mmol, and 6.37 ´ 108 mM·mg-1, respectively. Analysis of the hydrolysis products of N-acetyl chitooligosaccharides and colloidal chitin revealed that CmNAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)n+1 from (GlcNAc)n (n=1−6), respectively. In addition, CmNAGase also can catalyze GlcNAc to its dimers with various linked forms. Conclusions: The observations recorded in this study that CmNAGase is an exo NAGase with unique transglycosylation activity, suggests a possible application in the production of long-chain N-acetyl CHOs. This is first report of a bacterial NAGase, which can produce long-chain N-acetyl COSs via transglycosylation activity.

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

2020 ◽  
Author(s):  
Alei Zhang ◽  
Xiaofang Mo ◽  
Ning Zhou ◽  
Yingying Wang ◽  
Guoguang Wei ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Active Form ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Gene Encoding ◽  
Transglycosylation Activity ◽  
Reverse Hydrolysis ◽  
Hydrolysis Activity

Abstract 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.

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

2020 ◽  
Author(s):  
Alei Zhang ◽  
Xiaofang Mo ◽  
Ning Zhou ◽  
Yingying Wang ◽  
Guoguang Wei ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Active Form ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Gene Encoding ◽  
Transglycosylation Activity ◽  
Reverse Hydrolysis ◽  
Hydrolysis Activity

Abstract 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.

scholarly journals Cloning of the Novel Gene Encoding β-Agarase C from a Marine Bacterium, Vibrio sp. Strain PO-303, and Characterization of the Gene Product

2006 ◽  
Vol 72 (9) ◽  
pp. 6399-6401 ◽  
Author(s):  
Jinhua Dong ◽  
Shinnosuke Hashikawa ◽  
Takafumi Konishi ◽  
Yutaka Tamaru ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Gene Product ◽  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
The Novel ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Hydrolase Family

ABSTRACT The β-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. β-Agarase C was identified as the first β-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

scholarly journals Expression, purification and crystallization of a novel carbohydrate-binding module from theRuminococcus flavefacienscellulosome

2014 ◽  
Vol 70 (12) ◽  
pp. 1653-1656
Author(s):  
Immacolata Venditto ◽  
Maria S. J. Centeno ◽  
Luis M. A. Ferreira ◽  
Carlos M. G. A. Fontes ◽  
Shabir Najmudin
Keyword(s):  
Anaerobic Bacteria ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
The Novel ◽  
Carbohydrate Active Enzymes ◽  
Multiple Wavelength ◽  
Catalytic Module ◽  
Extensive Information ◽  
Hydrolase Family

Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing ofRuminococcus flavefaciensFD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. TheR. flavefaciensFD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin. The novel CBM-Rf1 has been purified and crystallized. The crystals belonged to the trigonal space groupR32:H. The CBM-Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment usingAutoSolfrom thePHENIXsuite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively.

scholarly journals Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 174 ◽  
Author(s):  
Manami Kobayashi ◽  
Yuya Kumagai ◽  
Yohei Yamamoto ◽  
Hajime Yasui ◽  
Hideki Kishimura
Keyword(s):  
Main Product ◽  
Specific Activity ◽  
Red Alga ◽  
Glycoside Hydrolase Family ◽  
Bifidobacterium Adolescentis ◽  
Glycoside Hydrolase Family 43 ◽  
Key Enzyme ◽  
Hydrolysis Of ◽  
Low Activity ◽  
Hydrolase Family

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

scholarly journals Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

Marine Drugs ◽  
2020 ◽  
Vol 18 (6) ◽  
pp. 312 ◽  
Author(s):  
Dandan Li ◽  
Shangyong Li ◽  
Yanhong Wu ◽  
Mengfei Jin ◽  
Yu Zhou ◽  
...  
Keyword(s):  
Dairy Products ◽  
Marine Bacterium ◽  
Intestinal Flora ◽  
Lactose Hydrolysis ◽  
Glycoside Hydrolase Family ◽  
Transglycosylation Activity ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Hydrolase Family

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. β-galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new β-galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 °C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0–9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

scholarly journals Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium acetobutylicum

2009 ◽  
Vol 76 (1) ◽  
pp. 338-346 ◽  
Author(s):  
Christopher D. Warner ◽  
Julie A. Hoy ◽  
Taran C. Shilling ◽  
Michael J. Linnen ◽  
Nathaniel D. Ginder ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Glycoside Hydrolase ◽  
Tertiary Structure ◽  
Nitrilotriacetic Acid ◽  
Product Distribution ◽  
Glycoside Hydrolase Family ◽  
Uncharacterized Protein ◽  
Gene Encoding ◽  
Tim Barrel ◽  
Hydrolase Family

ABSTRACT A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.

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