دورية أكاديمية
Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris.
العنوان: | 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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المؤلفون: | Pham TH; Institute of Biotechnology, Vietnam Academy of Science and Technology, Caugiay District 10600 Hanoi, Vietnam., Quyen DT, Nghiem NM, Vu TD |
المصدر: | Journal of microbiology and biotechnology [J Microbiol Biotechnol] 2011 Oct; Vol. 21 (10), pp. 1012-20. |
نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
اللغة: | English |
بيانات الدورية: | Publisher: Korean Society for Microbiology and Biotechnology Country of Publication: Korea (South) NLM ID: 9431852 Publication Model: Print Cited Medium: Internet ISSN: 1738-8872 (Electronic) Linking ISSN: 10177825 NLM ISO Abbreviation: J Microbiol Biotechnol Subsets: MEDLINE |
أسماء مطبوعة: | Publication: 2002- : Seoul : Korean Society for Microbiology and Biotechnology Original Publication: Seoul : Korean Society for Applied Microbiology, 1991-2001. |
مواضيع طبية MeSH: | Cloning, Molecular* , Gene Expression*, Aspergillus niger/*enzymology , Cellulase/*chemistry , Cellulase/*isolation & purification , Fungal Proteins/*chemistry , Fungal Proteins/*isolation & purification , Pichia/*genetics, Amino Acid Sequence ; Aspergillus niger/chemistry ; Aspergillus niger/genetics ; Base Sequence ; Cellulase/genetics ; Cellulase/metabolism ; Enzyme Stability ; Fungal Proteins/genetics ; Fungal Proteins/metabolism ; Kinetics ; Molecular Sequence Data ; Pichia/metabolism ; Substrate Specificity |
مستخلص: | 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 beta-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of 55°C and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of 30- 37°C 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 (K(m), V(max), k(cat), and k(cat)/ K(m)) determined for rEglA with beta-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 beta-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme. |
سلسلة جزيئية: | GENBANK GU445334 |
المشرفين على المادة: | 0 (Fungal Proteins) EC 3.2.1.4 (Cellulase) |
تواريخ الأحداث: | Date Created: 20111028 Date Completed: 20120207 Latest Revision: 20190923 |
رمز التحديث: | 20240628 |
DOI: | 10.4014/jmb.1104.04030 |
PMID: | 22031024 |
قاعدة البيانات: | MEDLINE |
تدمد: | 1738-8872 |
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DOI: | 10.4014/jmb.1104.04030 |