Study On Enzymatic Properties,Molecular Cloning And Food-grade Expression Of Chitosanase From Penicillium Oxalicum

Chitooligosaccharides have diverse biological activities and great application potential in the food field.As the most potential and valuable chitooligosaccharide production method,the specific enzymatic method has many advantages such as high efficiency,green control and so on.Chitosanase is the key craft in the enzymatic production of chitooligosaccharide.The existing chitosanases have some defects,such as low production efficiency,poor controllability to prepare chitosaccharides with high bioactivity and insufficient safety,which restrict their application in the food field.The purpose of this study was to explore new resources of chitosanase and apply them to the production of chitosaccharide with high efficiency and safety.The starting strains with high enzyme production capacity were screened from bird feces,and then the chitosanase produced by them was isolated and purified,and its enzymatic properties and types were analyzed.The nucleic acid sequence of the enzyme was cloned and heterologous expressed in Pichia pastoris GS115.The structural properties and catalytic mechanism of the enzyme were described by structural informatics analysis combined with site-directed mutagenesis.In order to expand the application prospect of this enzyme in food industry,a yeast-based chitosanase food-grade expression system was developed.The main research contents and results are as follows:（1）A total of 91 bird excrement samples were widely collected from important migratory bird habitats in the Yangtze River Basin,and strains with chitosan degradation ability were screened and identified by method with chitosan as the only carbon resource;CM-sepharose FF ion exchange chromatography was used to separate and purify chitosanase from crude enzyme solution,its enzymatic properties were studied,and the hydrolyzed products were analyzed by thin layer chromatography and high performance liquid chromatography;finally,the target protein was further identified by MALDI-TOF/MS.The results showed that among the 12enzyme-producing strains obtained in the preliminary screening,a filamentous fungus exhibited strong enzyme-producing ability（0.428 U/m L）.After morphological observation and ITS sequencing,it was identified as Penicillium oxalicum and named Penicillium oxalicum M2;two proteins with chitosan hydrolysis activity CSN1（42 k Da）and CSN2（110 k Da）were isolated from Penicillium oxalicum M2 fermentation supernatant by CM-sepharose FF ion exchange chromatography.Their optimum working conditions were p H 5.5,60°C and p H 5.0,60°C,respectively;CSN1 and CSN2 have better stability under the environment that p H is less than6,temperature is lower than 50°C and p H 4-8,temperature is lower than 55°C respectively;Thin-layer chromatography analysis showed that CSN 1 was a chitosanase with endo-hydrolysis of action.The main hydrolyzed products were chitobiose to chitopentaose and a small amount of hexasaccharide,and the proportion of tetra to hexasaccharide reached 61.91%.The specific activity of this enzyme reached 60.45 U/mg,which was the highest level of wild GH75 family chitanase reported.CSN 2 is a kind ofβ-D-glucosaminidase;Further investigation of the enzymatic properties of CSN1 revealed that Ca²⁺and Mn²⁺could significantly activate CSN1,while Zn²⁺,Fe²⁺and Cu²⁺significantly inhibited the hydrolytic activity of CSN1.In addition,different types of surfactants had an effect on the catalysis of CSN1,and non-ionic surfactants（Tweens 20/40/60/80 and Triton X-100）can enhance the hydrolysis effect of CSN1to different degrees.CSN1 was highly specific for chitosan substrates and tended to hydrolyze substrates with a high degree of deacetylation.The reaction kinetic parameters of CSN1 were K_m=0.54mg/m L,V_max=5.45μmol·m L^-1·min^-1.MALDI-TOF/MS identified the characteristic peptide in CSN1 as SALNKNYITNFSTLR,which was confirmed to belong to Conserved regions of multiple GH75 chitosanase family by BLASTP homology alignment in the NCBI protein database（www.ncbi.nlm.nih.gov/）（2）The full-length gene of chitosanase produced by P.oxalicum M2 was cloned to determine its amino acid sequence and properties.It was also exogenously expressed in Pichia pastoris GS115,and the enzymatic properties and hydrolysis patterns of the recombinase were studied.The results showed that the full-length nucleic acid sequence of the chitosanase was1041 bp encoding 346 amino acids,which was uploaded to Genbank and named Po CSN75A.Homology alignment and phylogenetic tree confirmed that it belongs to the GH75 chitosanase family;bioinformatics analysis predicted that the first 20 amino acids of its N-terminus were the wild signal peptide for auxiliary secretion;at the same time,the enzyme had a potential glycosylation site.PAS staining andβ-elimination reaction verified that it is an O-glycosylated protein;the secretion expression of Po CSN75A was successfully achieved in Pichia pastoris GS115,with an expression level of 5.09 mg/m L and an enzyme activity of 2.31 U.The enzymatic properties analysis of the recombinant chitosanase indicated that the enzymatic properties of the recombinant proteins were similar in all aspects,but the hydrolysis process was more severe,and the products were mainly chitobiose to chitotetraose.（3）In order to study the structural properties and catalytic mechanism of Po CSN75A,the prediction model of the target protein was constructed using the IDrug online server,the protein structure was optimized by molecular dynamics simulation.And then the key action sites,binding mode between enzyme and different substrates were predicted by molecular docking.The results showed that the key amino acid residues in the catalytic process were ASP148,GLU124,ASP150,THR149 and GLU159.The formation of hydrogen bonds between the hydroxyl group on the monosaccharide units of the substrate and the amino acid in the protein is its binding mode,and the binding ability of chitosanase to small molecular substrates increases with the increase of polymerization degree;The conserved carboxylic acid amino acid residues were mutated and analysis mutants by circular dichroism,it was proved that ASP148and GLU159 were the key residues for the enzyme to play a catalytic role.At the same time,a mutant CSN150（D150N）showed obvious increased activity,which is valuable for further study（4）The high specific activity mutant CSN150 obtained by site-directed mutagenesis was used as the research object,and analyzed its enzymatic properties;then,two mature GRAS strains,Saccharomyces cerevisiae and Pichia pastoris,were selected as expression hosts,using the two auxotrophs of URA3 and his4 as the screening markers,the compact expression fragment was designed to ensure the safety of the genetic tool,and the food-grade expression of the exogenous chitosanase mutant CSN150 was achieved by combining the endogenous P_GAPconstitutive promoter.The results showed that the enzymatic properties of CSN150 were consistent with the unmutated recombinase,but the hydrolyzed products were mainly chitotriose to chitohexaose in the middle and early stages,and chitobiose to chitopentaose as the major hydrolysate in the later stages of hydrolysis.In addition,HPLC analysis found that chitotetraose and chitopentaose accounted for 56.4%of the final hydrolysis products;The antibody-free compact fragments corresponding to Saccharomyces cerevisiae and Pichia pastoris were integrated into the host,and the expression of CSN150 in Saccharomyces cerevisiae was extremely low,while Pichia pastoris showed better CSN150 secretion and expression;The optimal fermentation conditions were 28°C,p H 5.5 and glucose as carbon source,and the maximum enzyme production capacity could be achieved（2.08 U/m L）after fermentation for 60 h.In conclusion,avian gut microbes can be used as a source for screening chitosanase-producing strains.The specific activity of chitosanase produced by Penicillium oxalicum M2was higher than that of most wild-type fungal chitosanase.Its enzymatic properties and hydrolysis patterns indicate that it has certain potential in the production of chitotetraose to chitopentaose.At the same time,the structural properties,catalytic mechanism and suitable exogenous expression host of the enzyme were studied by molecular biology techniques.And developed a chitosanase food-grade expression engineering microorganism based on Pichia pastoris,which significantly improved the efficiency and safety of enzyme production.This study enriched the specific chitosanase resources for chitosaccharide production and explored a new strategy for chitosaccharide production with high efficiency and safety,which is of great significance for the safe application of chitosaccharide in food industry.