The Rational Design Of Pectate Lycase PEL168 And Efficient Expression In Pichia Pastoris

Pectinase are a group of enzymes that catalyze pectin.According to its function of the optimum pH,pectinase can be divided into acidic pectinase and alkaline pectinase.Acidic pectinase is widely used in extraction ofjuices or wines,and feed industry.Alkaline pectinase gets more attention in oil extraction,plant medicine extraction,plant fiber processing,textile,paper making,detergent coffee and tea fermentation,industrial wastewater treatment and the application of bio logical technology of pectin.So it becomes the research hot spot.In industrial application,because of the narrow suitable range of pH,we need more technolo gy to maintain the stability of enzyme in the reaction system,meanwhile need a large amount of acid or alkali to neutralize,which will have the high costs,the water consumpotion,the large energy consumption,the time consuming,the high COD in waste water.At the same time,the efficiency and versatility of pectinase research is imminent.So it is the key to get the pectinase of more efficient and more substrate specificity,more stable enzymatic properties in the research development of pectinase.The early study was obtained the alkaline pectate lyase pel168 genes from the Bacillus subtilis 168 by PCR technology.The gene had the ORF containing 1263 bp,and encoded 420 amino acids.Using the Signal P,the amino acid sequence was consisted of 21-residue signal peptide.The gene pel168 was expressed in the E.coli Rosset Blue(DE3),then the enzymatic properties of the enzyme have been characterized after it was purified.The results showed that the optimum temperature of the alkaline specific activity was 50? and the optimum pH was 9.4.However,the specific activity of the PEL168 was not high,and the relative activity also was reduced by 40%when the pH was 3?6.Therefore,this research carry on the rational design of PEL168,use the PyMol of auxiliary software analyze the tertiary structure of the pectate lycase PEL168,and estimate the three plans:1)mutation the 26th K and 27th A into E and D respectively,and the aim is form salt bridge with the 5th H to increase the terminal compactness;2)mutation 132th V into the amino acid with a benzene ring to enhance the hydrophobicity;3)on the basis of the second plan,mutation 3th L into the amino acid with a benzene ring to do double mutation expression analysis.Then design the primers for site-directed mutagenesis,study the mutant protein on its structure and function.In the first plan,we mutate the 26th K and 27th A into E and D respectively,obtain mutant,study on the enzyme activity,found that the mutant completely loss of enzyme activity;In the third plan,we mutate 3th L into phenylalanine F when on the basis of the second plan,obtain mutant,study on the enzyme activity,found that compared with the original protein,the enzyme activity of the mutant greatly reduced;Because of in the first and third mutation scheme,compared with the original protein,the enzymology properties of the mutants have not been improved,and for the second scheme,turn the genes of a 457-bit guanine G into thymine T,obtain pe1168V132F,puts the peptide 132th valine V(GTC)mutation into phenylalanine F(TTC)of its encoding amino acid sequence.When respectively putting the original gene pe1168 and the mutation gene pell68 V132F to induce expression in E.coli Rosset Blue(DE3),then purifying the expression product for nickel column affinity chromatography and fast protein liquid chromatography(FPLC),finally comparing the enzymatic properties of PEL168 with the enzymatic properties of pel168V132F,we find that the relative activity of pe1168V132F has increased significantly comparing with the original pel1 68 when pH is 3?5?6,at the same time the specific activity is 2.25 times higher than the original one.In addition,in order to improve the expression level of the enzyme in the yeast,the expression vector for the optimized gene pel168s was changed basing on the codon preference and degeneracy of the Pichia pastoris.And we convert the original carrier pHBM905A to the plasmid vector BDM with stronger signal peptide and promoter,construct recombinant plasmid BDM-pell68s into Pichia pastoris GS115,and induce the expression of heterologous protein(PEL168S).After selecting the Pichia pastoris recombinant strains with the same copy numbers before and after the change,inducing 96 h by methanol under the condition of the same culture,we find that the enzyme activity of BDM-PEL68S has increased by 47%comparing with pHBM905A-PEL168S.The result of fluorescence quantitative PCR technology in the mRNA level shows that the mRNA expression level after replacing the expression vector of recombinant strains increases by 27%than the original.