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Enzymatic production of glucosamine and chitooligosaccharides using newly isolated exo-?-D-glucosaminidase having transglycosylation activity.

ABSTRACT: Exochitosanase secreting fungus (A. fumigatus IIT-004) was isolated from fish waste using 1 % (w/v) chitosan as sole carbon source after multistage screening. Chitosan-dependent exochitosanase enzyme production (6 IU ml-1) in log phase of growth (chitosan utilization rate 0.11 g g-1 cell h-1) was observed for Aspergillus fumigatus in chitosan minimal salt medium and there was no enzyme production in glucose medium. Enzyme production was found to be extracellular and subjected to purification by a number of steps like acetone fractionation as well as column chromatography. 40 % yield and 26-fold of enzyme purification was achieved after all the steps. Purified enzyme was characterized for optimum temperature, pH, ionic strength and substrate specificity. The K m and V max for purified exochitosanase enzyme was calculated to be 8 mg ml-1 and 5.2 × 10-6 mol mg-1 min-1. Enzyme was immobilized on polyacrylonitrile nanofibres membrane matrix by adsorption as well as amidination. Enzymatic production of glucosamine was achieved using various chitosan substrates by free/immobilized exochitosanase and compared. Isolated and purified exochitosanase also showed transglycosylation activity.

SUBMITTER: Sinha S

PROVIDER: S-EPMC4703589 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Enzymatic production of glucosamine and chitooligosaccharides using newly isolated exo-β-D-glucosaminidase having transglycosylation activity.

Sinha Sujata S Chand Subhash S Tripathi Pushplata P

3 Biotech 20160106 1

Exochitosanase secreting fungus (A. fumigatus IIT-004) was isolated from fish waste using 1 % (w/v) chitosan as sole carbon source after multistage screening. Chitosan-dependent exochitosanase enzyme production (6 IU ml-1) in log phase of growth (chitosan utilization rate 0.11 g g-1 cell h-1) was observed for Aspergillus fumigatus in chitosan minimal salt medium and there was no enzyme production in glucose medium. Enzyme production was found to be extracellular ...[more]

PMID: 28330083

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Project description: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 toward natural killer cells. Thus, it is of great significance to discover a ?-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. Results:The gene encoding the novel ?-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 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m of CmNAGase for pNP-GlcNAc were 16,666.67 ?mol min-1 mg-1, 0.50 ?mol mL-1, 25,555.56 s-1, and 51,111.12 mL ?mol-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, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Project description:Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages (teji, tella, shamiata and areqe tinisis), milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 10 5 CFU from traditional foods and beverages to that of 10 3 CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates (ETP37, ETP50; ETP53, ETP89, ETP94) were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34 o C. pH 4.8, temperature 36 o C, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production.

Dataset Information

Enzymatic production of glucosamine and chitooligosaccharides using newly isolated exo-?-D-glucosaminidase having transglycosylation activity.

Publications

Enzymatic production of glucosamine and chitooligosaccharides using newly isolated exo-β-D-glucosaminidase having transglycosylation activity.

Similar Datasets

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