title: Production of Chimeric Acidic α-Amylase by the RecombinantPichia pastorisand Its Applications.
abstract: Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly inPichia pastorisunderAOXpromoter. Clones ofP. pastoriswith 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 recombinantP. pastorisintegrated eight copies ofBa-Gt-amyin the genome ofP. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level ofBa-Gt-amywas higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed inP. pastorisis a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2of 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.
authors.affiliation: Department of Microbiology, University of Delhi New Delhi, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Parashar
authors.affiliation: Department of Microbiology, University of Delhi New Delhi, India.
authors.forename: Tulasi
authors.initials: T
authors.lastname: Satyanarayana
keywords: Pichia pastoris; chimeric α-amylase; multi-copy clones; post-transformational vector amplification; starch saccharification