Dataset Information

Purification, characterization, and statistical optimization of a thermostable ?-amylase from desert actinobacterium Streptomyces fragilis DA7-7.

ABSTRACT: In this study, preliminary screening revealed that of 134 desert soil actinobacterial isolates, only 43 isolates produced amylase. Among these, an isolate DA7-7, which was identified as Streptomyces fragilis DA7-7, showed a prominent zone of clearance and significant amount of ?-amylase production. The pre-optimization studies showed varying physicochemical and nutrients properties of the medium influenced the enzyme production significantly. Consequently, central composite design was employed with the selected variables (pH, temperature, dextrose, and peptone) for ?-amylase production. The optimum fermentation conditions were 3.07% dextrose, 1.085% peptone, pH 6.0, and incubation temperature 27.27 °C. The predicted optimum ?-amylase activity was 991.82 U/mL/min, which was similar to the experimental amylase activity of 973.5 U/mL/min. The crude ?-amylase produced by S. fragilis DA7-7 was purified with ammonium sulfate precipitation, followed by gel filtration chromatography, and the estimated molecular mass was 51 kDa. The purified ?-amylase was stable under the following conditions: pH (4-9), temperature (40-80 °C), NaCl (1-4 M), and detergents (1-10 mM). The Km and Vmax values of enzyme were found to be 0.624 mU/mg and 0.836 mg/mL, respectively.

SUBMITTER: Nithya K

PROVIDER: S-EPMC5614901 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Purification, characterization, and statistical optimization of a thermostable α-amylase from desert actinobacterium <i>Streptomyces fragilis</i> DA7-7.

Nithya Krishnasamy K Muthukumar Chinnasamy C Kadaikunnan Shine S Alharbi Naiyf S NS Khaled Jamal M JM Dhanasekaran Dharumadurai D

3 Biotech 20170926 5

In this study, preliminary screening revealed that of 134 desert soil actinobacterial isolates, only 43 isolates produced amylase. Among these, an isolate DA7-7, which was identified as <i>Streptomyces fragilis</i> DA7-7, showed a prominent zone of clearance and significant amount of α-amylase production. The pre-optimization studies showed varying physicochemical and nutrients properties of the medium influenced the enzyme production significantly. Consequently, central composite design was emp ...[more]

PMID: 28955647

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Project description:BackgroundChinese Nong-flavor (NF) liquor is continuously and stably produced by solid-state fermentation technology for 1000 years, resulting in enrichment of special microbial community and enzymes system in its starter. Based on traditional culture-dependent methods, these functional enzymes are hardly obtained. According to our previous metatranscriptomic analysis, which identifies plenty of thermostable carbohydrate-active enzymes in NF liquor starter, the aim of this study is to provide a direct and efficient way to mine these thermostable enzymes.ResultsIn present study, an alpha-amylase (NFAmy13A) gene, which showed the highest expression level of enzymes in starch degradation at high temperature stage (62 °C), was directly obtained by functional metatranscriptomics from Chinese Nong-flavor liquor starter and expressed in Pichia pastoris. NFAmy13A had a typical signal peptide and shared the highest sequence identity of 64% with α-amylase from Aspergillus niger. The recombinant enzyme of NFAmy13A showed an optimal pH at 5.0-5.5 and optimal temperature at 60 °C. NFAmy13A was activated and stabilized by Ca2+, and its half-lives at 60 and 70 °C were improved significantly from 1.5 and 0.4 h to 16 and 0.7 h, respectively, in the presence of 10 mM CaCl2. Meanwhile, Hg2+, Co2+ and SDS largely inhibited its activity. NFAmy13A showed the maximum activity on amylopectin, followed by various starches, amylose, glycogen, and pullulan, and its specificity activity on amylopectin was 200.4 U/mg. Moreover, this α-amylase efficiently hydrolyzed starches (from corn, wheat, and potato) at high concentrations up to 15 mg/ml.ConclusionsThis study provides a direct way to mine active enzymes from man-made environment of NF liquor starter, by which a fungal thermostable α-amylase (NFAmy13A) is successfully obtained. The good characteristics of NFAmy13A in degrading starch at high temperature are consistent with its pivotal role in solid-state fermentation of NF liquor brewing. This work would stimulate mining more enzymes from NF liquor starter and studying their potentially synergistic roles in NF liquor brewing, thus paving the way toward the optimization of liquor production and improvement of liquor quality in future.

Dataset Information

Purification, characterization, and statistical optimization of a thermostable ?-amylase from desert actinobacterium Streptomyces fragilis DA7-7.

Publications

Purification, characterization, and statistical optimization of a thermostable α-amylase from desert actinobacterium <i>Streptomyces fragilis</i> DA7-7.

Similar Datasets

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