Beta-Glucosidase From Thermotolerant Yeast Pichia Etchellsii: Gene Sequencing, Cloning And Functional Expression In Methylotrophic Yeast Pichia Pastoris
Journal Title: INTERNATIONAL JOURNAL OF CURRENT RESEARCH - Year 2016, Vol 8, Issue 2
Abstract
Pichia etchellsii Bgl1 gene coding for BGLI protein was fished out from yeast genomic DNA using PCR based strategies. The primers were designed based on the internal peptide sequences of native BGLI protein. Bgl1 gene was cloned and expressed in Pichia pastoris. The deduced amino acids encoded by Bgl1 showed high similarity with the sequences of Glycoside hydrolase family 3 members. The predicted isoelectric point (pI) of the protein was 5.2 and A+T% and G+C% were 58.06% and 41.95% respectively. The multiple sequence alignment using ClustalV program of DNASTAR software showed 98.6 % identity with a hypothetical 765 aa protein of Kluyveromyces lactis and 73.4% with 845 aa β-glucosidase protein of Kluyveromyces fragilis. The recombinant enzyme showed maximal activity at pH 6.0 and was stable between pH 3.5–9. More than 80% of enzyme activity was retained in this pH range on incubating enzyme for 24 h. The enzyme had temperature optimum of 50 °C under optimal pH with pNPG as substrate.
Authors and Affiliations
Dr. Richa Baranwal
Keywords
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