Improving The Thermostability Of Alkaline Polygalacturonate Lyase By N-glycosylation Modification

Alkaline polygalacturonate lyase,a polygalacturonase,can break off the ?-1,4 glycoside of polygalacturonic acid by trans elimination under alkaline conditions(p H8~11)and generate unsaturated oligogalacturonic acid.PGL is a kind of industrial enzyme with very high commercial value because it is widely used in the degradation of pectin in the fields of textile,environment,papermaking and food.It Alkaline polygalacturonate lyase is widely distributed in microorganisms,but the ability of wild type strains to produce alkaline polygalacturonate lyase is generally limited,which seriously limits the large-scale production of alkaline polygalacturonate lyase.In recent years,researchers have begun to construct recombinant bacteria using genetic engineering techniques.Recombinant alkaline polygalacturonate lyase has been successfully expressed in Escherichia coli,Bacillus subtilis and Pichia pastoris.Pichia pastoris has the advantages of genetic stability,strong secretion system,easy high-density culture,low expression of extracellular protein and other advantages become one of the most talked about expression of host.However,previous studies mainly focused on the construction of recombinant bacteria and the optimization of fermentation conditions.There are not many researches on the transformation of PGL from the gene level to improve its expression efficiency and improve its enzymatic properties.It is very few that involves the research of N-glycosylation.In our previous work,we found that introducting alkaline polygalacturonate lyase gene into Pichia pastoris resulted in the N-glycosylation of this enzyme at three sites including 128,185 and 353.Thereupon,the N-glycosylation modification of the recombinase was started.The asparagine at these three sites was mutated to glutamine,respectively,and combined mutation was performed to obtain a recombinant strain GS115/N185Q-N353 Q with high activity but poor thermostability.In this study,alkaline polygalacturonate lyase was further N-glycosylated by site-directed mutagenesis in order to obtain mutant enzymes with high thermostable stability.By analyzing the results of previous experiments,we hypothesized that the removal of N-glycosylation would adversely affect the thermal stability of PGL probably because some of the N-glycosylated asparagines were replaced by glutamine Destroy the original force between the surrounding amino acids and thus change the regional structure of the protein,resulting in higher energy after mutation.If the conversion mode removes N-glycosylation,for example,asparagine is mutated to a lower amino acid than glutamine,or the asparagine is not mutated but the subsequent threonine is mutated to a serine Amino acids with lower external mutation may reduce the adverse effects on thermal stability.This is the first strategy to modify the thermal stability of alkaline polygalacturonate lyase by N-glycosylation in this experiment.In addition,many reports report that the introduction of N-glycosylation enhances the thermostability of enzymes,but this effect is point-specific.Recent studies have shown that the insertion of a new N-glycosylation with an enhanced aromatic sequence profile in the reverse corner of the protein can more reliably increase its thermal stability.The amino acid composition of enhanced aromatic sequence is in three forms: F-N-X-T,F-X-N-X-T and F-X-X-N-X-T(F is phenylalanine,N is asparagine,X is any amino acid other than proline and T is threonine.Compared to the normal N-glycosylation sequence NXT,an aromatic amino acid phenylalanine is present at the 1~3 positions in front of the asparagine of enhanced aromatic sequence,and phenylalanine,when located in the reverse-angled region,Aspartame-linked sugar chains and threonine interact and form a stable structure in the region of the module,which contribute to the stability of the overall protein conformation.Accordingly,we can screen the reverse corner of alkaline polygalacturonate lyase to introduce enhanced aromatic sequence to further improve its thermal stability,which is the second strategy to modify the thermal stability of alkaline polygalacturonate lyase in this experiment.Based on the software modeling and analysis,through the first strategy,the half life of alkaline polygalacturonate lyase has been increased by 176%.Although it is far from the target value,some achievements have been achieved as the first phase of modification.Then,we selected three suitable sites to be introduced into the new N-glycosylation with enhanced aromatic sequence sequence characteristics respectively,followed by combined mutagenesis.The half-life of alkaline polygalacturonate lyase was further increased to 1.35 h,close to the target value.These two strategies not only did not negatively affect the catalytic activity of the enzyme,but increased the specific activity slightly,that is,we obtained alkaline polygalacturonate lyase with better enzymatic properties in Pichia pastoris.This study shows that it is of interest to mutate the amino acid sequence of this site to determine the optimal mutation before de-N-glycosylation;to introduce a new N-glycosylation with enhanced aromatic sequence sequence features in the reverse-turn region of the enzyme To improve its thermal stability has a significant role in promoting.These conclusions have certain referential significance for the N-glycosylation of recombinase in Pichia pastoris.