Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 104, Issue -, Pages 107-116Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.ijbiomac.2017.05.167
Keywords
Codon optimization; Constitutive expression; Thermophilic mould; Myceliophthora thermophila; Thermo-alkali-stable endoglucanase; Enzymatic saccharification
Funding
- Indo-US Science & Technology Forum (IUSSTF), University Grants Commission, Govt. of India, New Delhi
- University of Delhi
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Codon adaptation index (CAI) of a 1263 bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of 47 kDa. rMt-egl is optimally active at pH 10 and 50 degrees C, displaying stability in broad pH and temperature ranges, with a t(1/2) of 60 and 15 min at similar to 90 and 100 degrees C, respectively. This retained similar to 70% of activity after 3 h incubation at pH 5-12. The K-m, V-max, K-cat and k(cat)/K-m of rMt-egl were 5 mg mL(-1), 20 mu moles min(-1) mg(-1), 1.02 x 10(3) s(-1) and 204s(-1) mg(-1) mL(-1), respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382 mg g(-1)), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues. (C) 2017 Elsevier B.V. All rights reserved.
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