期刊
FRONTIERS IN MICROBIOLOGY
卷 8, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2017.00493
关键词
chimeric alpha-amylase; Pichia pastoris; multi-copy clones; post-transformational vector amplification; starch saccharification
类别
资金
- Department of Science and Technology (DST), Government of India, New Delhi [SR/SO/BB-109/2010]
Recombinant chimeric alpha-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in alpha-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60 degrees C with T-1/2 of 40 min at 70 degrees C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.
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