Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris

Abstract

Pectinase is one of the most widely used enzymes in different fields of food industry for different purposes, such as clarification of fruit juice, extraction of vegetable oil and saccharification of agricultural substrates. The aim of this study is to produce recombinant pectinase and evaluate the effect of codon optimization and promoter selection on production.In this study, the gene encoding pectinase in Aspergillus niger was optimized according to the codon usage of Pichia pastoris. Within the scope of the study, codon-optimized and native (non-codon-optimized) pectinase genes were transferred to P. pastoris X33 strain and expressed under the regulation of methanol-inducible AOX1 and ethanol-inducible ADH2 promoter.As a result of the study, the promoter and codon combination which exhibited the highest production level was determined as the ADH2 and codon-optimized pectinase yielding an enzyme activity of 42.33 U/mL. The best producer clone was cultured in 400 mL media in 2 L shake-flask and the enzyme was purified by His-tag method. The optimum working conditions of the purified enzyme was 50 °C and pH 5.0, and after incubation at 60 °C for 1 -h, enzyme activity was maintained at 60% level. The Michelis–Menten constant (Km) and maximal velocity (Vmax) of the purified recombinant pectinase were 6.9 mg/mL and 67.57 μmol/mg/min, respectively.