Expression Of Pullulanase In Pichia Pastoris And Computational-based Site-directed Mutagenesis

The pullulanase was widely appied in the starch industry, as it can hydrolyze the α-1,6-glucosidic bond of amylopectin which leads the low utilization rate of amylum.In this study, the gene from Bacillus acidopullulyticus was used to express in Pichia pastoris. And then the recombinant pullulanase was modified with site-directed mutagenesis by computational design. The research mainly focuses on the following aspects:(1) Codon optimization, gene synthesis, expression and characteristics of the recombinant Pul13The pullulanase gene was optimized according to the codon usage bias. The full-length gene was obtained by assembling these fragments by dual asymmetric PCR and overlap extension PCR and then was electrotransformated into Pichia pastoris-X33.The optimum temperature and pH of restructuring pullulanase were60℃and4.0-5.0, respectively. When the temperature was below60℃, its activity could keep more than70%of the highest enzyme activity. But there was a sharp decline in enzyme activity when above60℃. When pH was less than2.0or above7.0, the enzyme activity was almost lost. After treatment in different pH buffer under4℃for1h, the result showed it was well stability from pH3.0-6.0. Ca2+, Mg2+, Fe3+and Fe2+with the concentration of1mM had certain promoting effect to enzyme activity and Na+and K+had no obvious effect on enzyme activity. But,1mM EDTA, Cu2+, Ba2+, SDS and50mM Na+and K+could inhibit the enzyme activity. Taking pullulan polysaccharides as the substrate, the Michaelis constant (Km) of recombinant enzymes was0.26mg/mL and its maximum reaction rate (Vmax) was2.32μmol·min-1·mg-1.(2) Selection and construction of mutation sites of pullulanase.Based on the analysis of3D structure of pullulanase,5loops which were closed with the catalytic pocket were chosen as the mutation sites. These loops were changed into the corresponding amino acids of the pullulanase from Klebsiella pneumonia to study the influence of amino acid in each loop to the specific activity. In addition, glutamine in the site of487and the half-conserved asparagine in the site of495were chosen to mutate into alanine. The overlap extension PCR was used to obtain the mutant gene sequence and finally seven mutants had been successfully expressed in Pichia pastoris X-33.(3) Properties analysis of recombinant enzyme with site-directed mutagenesisCompared with the properties of the wild type pullulanase and the mutations, the specific activity of Pul13M1had increased from2.21U/mg to3.22U/mg, and the optimal temperature also shifted from60℃to75℃. The activity of pullulanase Pul13M2also increased to2.96U/mg. The specific activities of the other mutant strains were decreased. The optimal pH of pullulanase Pul13M45and Pul13M7also changed from4.0-5.0to6.0.This research took the site-directed mutagenesis by computational design to change the enzymology properties of pullulanase. It could lay the foundation for subsequent modification of enzyme properties.