AUTHOR=Parashar Deepak , Satyanarayana Tulasi 

TITLE=Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.00493

DOI=10.3389/fmicb.2017.00493

ISSN=1664-302X

ABSTRACT=<p>Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in <italic>Pichia pastoris</italic> under <italic>AOX</italic> promoter. Clones of <italic>P. pastoris</italic> with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant <italic>P. pastoris</italic> integrated eight copies of <italic>Ba-Gt-amy</italic> in the genome of <italic>P. pastoris</italic>, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of <italic>Ba-Gt-amy</italic> was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in <italic>P. pastoris</italic> is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T<sub>1/2</sub> of 40 min at 70°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.</p>