Fungal Thermophilic Polygalacturonase Gene Exploit And Catalytic Efficiency Molecular Modification

Pectinase is widely applied to juice clarification of food industry as additive.Finding and mining quality pectinase suitable for industrial application offers more options for industrial application.In this study,three polygalacturonase were cloned from thermophile Talaromyces leycettanus JCM 12802,named Tepg28 a,Tepg28b and Tepg28 c,one exopolygalacturonase and the other two endopolygalacturonase,all of which were in high expression in Pichia pastoris GS115.We studied the enzymatic properties of the three recombinant proteins after purification,under optimal pH 3.5 and optimal temperature 70 oC.All three showed good PH stability in acidic and neutral environment,and high stability under 60 oC,and there was 40% residual activity at 70 oC for 10 minutes.Note that the specific activity of TePG28 c reached 51786 U/mg,higher than all reported polygalacturonase.TePG28 a and TePG28 b are two different types of polygalacturonase with different catalysis manners.The combination in ratio of 2:3(TePG28a: TePG28 b = U:U)showed good synergistic effect for orange peel pectin,240% higher efficiency than either one,and both together showed synergistic effect for grape juice clarification,increasing light transmittance of grape juice from 20% to over 80%.In general,the three polygalacturonase own favorable enzymatic enzymatic properties,showing huge potential industrial application.Based on above,this study chose TePG28 b as the subject.According to the analysis of the protein structure and homologous alignment,and the transformation strategy of the introduction of positively charged amino acids to non-binding site of substrate binding region,catalytic efficiency improvement was expected by enhancing the binding of the substrate to the enzyme-catalyzed pocket.The result showed that the strategy had an obvious effect for TePG28 b.Compared with the soluble enzyme,TePG28bD113 K mutant showed looser substrate affinity,but 80% higher specific activity and 20% higher catalytic efficiency.This transformation strategy was also verified from endopolygalacturonase BsPG28 a cloned from Bispora sp.MEY-1.Compared with the soluble enzyme BsPG28 a,BsPG28aD107K mutant showed same substrate affinity but 280% higher specific activity and 250% higher catalytic efficiency.Both mutants,compared with soluble ones,showed improvement on catalytic efficiency and specific activity,and the improvement of BsPG28aD107 K was more obvious than TePG28bD113 K.In short,in this study,three quality polygalacturonase of good enzymatic properties were cloned from thermophilic fungus Talaromyces leycettanus JCM 12802,showing huge application potential after researches on the enzymatic properties,synergistic effect and application valuation,which provided choices and reference for industrial enzymes.The introduction of positively charged amino acids to non-binding site mutation of substrate binding region of polygalacturonase increased specific activity and catalytic efficiency of TePG28bD113 K and BsPG28 a,which provided an effective strategy for catalytic efficiency molecular modification of polygalacturonase,and showed important scientific significance and application value.