Secretion Expression Of Maltotetraose-Forming Amylase From Pseudomonas Saccharophila And Analysis Of Its Structure And Enzymatic Properties

Maltooligosaccharides,novel functional oligosaccharides composed of 3 to 10α-D-glucopyranoses linked byα-1,4-glycosidic bonds,have favorable physiochemical properties and special physiological function,thus leading to the profound application in food and medicine industry.The maltooligosaccharide-forming amylases（MFAses）are main enzyme preparations for maltooligosaccharides production.With the deep researches at home and abroad,several MFAses derived from different species have been heterologously expressed but with low secretion levels,restricting their industrial application.Moreover,studies on the structure-function relationship and catalytic mechanism of MFAses are limited,which are quite essential for deeper research and molecular modification.This study focused on the MFAse from Pseudomonas saccharophila STB07(MFA_ps).Firstly,the Escherichia coli BL21（DE3）,Bacillus subtilis WB600 and Pichia pastoris GS115 expression systems were established,and secretory expressions of MFA_ps were proceeded.The results showed that B.subtilis WB600 was the optimal expression system for MFA_ps,with the maximum crude activity being 144.60 U/mL which were 13.7 times higher than that reported before and the specific activity being 980.49 U/mg.In comparison,MFA_ps in E.coli BL21（DE3）showed a much lower crude activity of 6.49 U/mL after 72 h and the amylase in P.pastoris GS115 had a sharply decreased specific activity of 220.21U/mg despite its high secretion level of 225.23 U/mL after 72 h.Secondly,the enzymatic properties of MFA_ps in B.subtilis WB600 were determined.It showed that the optimal reaction temperature and pH were 50⁵5oC and pH 6.5,respectively.MFA_ps exhibited excellent thermostability in pH 8.5 Gly-NaOH buffer,with 88%remnant activity after incubation for 120 min at 50oC.Furthermore,5 mmol/L Ca²⁺could enhance the thermostability,with over 80%activity detected after incubation in pH 7.0 Tris-HCl buffer for 2 h.The dynamics properties were measured via hydrolysis of different substrates.Results showed that MFA_ps had much higher affinity and catalytic sufficiency toward amylopectin than amylose,and among maltodextrin and other starch（soluble starch,maize starch,waxy maize starch,potato starch,tapioca starch and wheat starch）,waxy maize starch could be hydrolyzed with higher affinity and catalytic sufficiency than the rest substrates.In addition,the action pattern of MFA_ps were inclined to be exo-type.In order to study on the catalytic mechanism of MFA_ps,its structure was obtained at 1.5?,indicating 3 domains containing a（β/α）₈ barral structure and 2 Ca²⁺binding sites.Subsequently,the co-crystal structure of MFA_ps/G4 and MFA_ps/pG7 were determined at 1.5?and 1.1?,respectively,revealing subsites-4⁺3 in the catalytic core.We presented evidence that Gly158 and Glu160 and hydrophobic stacking are essential for substrate binding.Furthermore,product specificity is influenced by both the number of active cleft subsites and the endo-/exo-type action pattern,and strong substrate interactions with two loop structures may govern G4 specificity help determine the exo-type action pattern of MFA_ps.Whereafter,the product specificity of MFA_ps were proved using HPAEC-PAD on the basis of analysis on its action pattern and structures.Results showed that MFA_ps could produce G4 in absolute predominance on the degradation of maltooligosaccharides with DP>4 as well as amylose and amylopectin.Notably,the percentage of G4 reached to 76.25%on hydrolysis of amylopectin and the conversion rate was 72.27%on amylose.Finally,the reaction conditions for MFA_ps producing G4 were optimized as follows:2%maize starch solution（pH 8.5 Gly-NaOH buffer）was hydrolyzed by 2 U/g MFA_ps at 50oC for 48 h,with the conversion rate and G4 percentage being 72.21%and 76.75%,respectively.