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KMID : 0545120110210101012
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Journal of Microbiology and Biotechnology
2011 Volume.21 No. 10 p.1012 ~ p.1020
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Cloning, Expression, Purification, and Properties of an Endoglucanase Gene (Glycosyl Hydrolase Family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris
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Thi Hoa Pham
Dinh Thi Quyen
Ngoc Minh Nghiem
Thu Doan Vu
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Abstract
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A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of β-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of 55oC and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of 30- 37oC and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants (Km, Vmax, kcat, and kcat/ Km) determined for rEglA with β-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 min-1, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 min-1, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward β-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.
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KEYWORD
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Aspergillus niger VTCC-F021, Endoglucanase gene, Cloning, Expression, Properties
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