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

Abstract

Recombinant chimeric α-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 α-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°C with T1/2 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.

Characterization of Full-Length and Truncated Recombinant κ-Carrageenase Expressed in Pichia pastoris

“…The target genes cgkZ and cgkZ Δ Pst had the same copy numbers. It was reported that gene copy number and heterologous protein expression had a linear correlation ( Parashar and Satyanarayana, 2017 ). From the results of shaker-flask fermentation and purification, cgkZΔPst showed a 1.2-fold higher enzymatic activity and 1.4-fold higher specific activity than cgkZ.…”

An Insight Into Ameliorating Production, Catalytic Efficiency, Thermostability and Starch Saccharification of Acid-Stable α-Amylases From Acidophiles

“…The recombinant acid-stable Ba-amy purified from P. pastoris was biochemically characterized, which revealed kinetic properties and thermostability of glycosylated acid-stable Ba-amy to be similar to those of the recombinant acid-stable Ba-amy expressed in E. coli . The engineered Ba-amy (Ba-Gt-amy) was also cloned and expressed in P. pastoris (Parashar and Satyanarayana, 2017a ). The combination of multiple transformations and post-transformational vector amplification (PTVA) and high cell density cultivation in fermentor led to a very high production (750 U/mL) of the chimeric Ba-Gt-amy (Parashar and Satyanarayana, 2017a ).…”

“…The engineered Ba-amy (Ba-Gt-amy) was also cloned and expressed in P. pastoris (Parashar and Satyanarayana, 2017a ). The combination of multiple transformations and post-transformational vector amplification (PTVA) and high cell density cultivation in fermentor led to a very high production (750 U/mL) of the chimeric Ba-Gt-amy (Parashar and Satyanarayana, 2017a ).…”

“…In the first step, amylase was engineered by adding 11 and 37 amino acids to N- and C- terminal ends from the α-amylase of G. thermoleovorans . In the second attempt, the engineered amylase and glucoamylase (from Aspergillus niger ) were fused through a linker peptide of 25 amino acids [(Gly-Gly-Thr-Gly-Ser) 5 ] {(Gly-Gly-Thr-Gly-Ser) 5 } (modified with permission from Parashar and Satyanarayana, 2016c , 2017a ).…”

Recapitulation of stability diversity of microbial α-amylases

“…Compared to wild type, the recombinant α-amylase produced extracellularly in P. pastoris showed 10.7-fold times improved activity and the expressed enzyme exhibited optimal activity at pH 4.0 and 60 °C. Since the enzyme could saccharify both soluble and raw starch and release maltose, it is used in baking and sugar syrup industries [76].…”