Dataset Information

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 ?-Glucanase

ABSTRACT: Background:Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of ?-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods:bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents. Results:The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors. Conclusion:Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.

SUBMITTER: Rezaie M

PROVIDER: S-EPMC6058188 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase

Rezaie Meysam M Aminzadeh Saeed S Heidari Farid F Mashhadi Akbar Boojar Masoud M Karkhane Ali Asghar AA

Iranian biomedical journal 20180113 5

<h4>Background</h4>Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01.<h4>Methods</h4>bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was pur ...[more]

PMID: 29331014

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Project description:BACKGROUND: ?-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ?-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ?-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ?-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ?-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the ?-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes ?-1,3-glucan. Insoluble ?-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ?-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble ?-1,3-glucan, and could be classified into CBM families.

Dataset Information

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 ?-Glucanase

Publications

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase

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