Secretion Expression Of Maltooligosaccharide-forming Amylase In Bacillus Subtilis And Characterization Of Its Enzymatic Properties And Product Synthesis

Maltooligosaccharides are functional oligosaccharides composed of 3 to 10α-D-glucopyranosyl units linked byα-1,4-glycosidic linkages,and they show great prospects in food,medicine,chemical industries and so on.They can be produced by using maltooligosaccharide-forming amylases（MFAses）to hydrolyze starch.Nowadays,their industrial production is monopolized by some developed countries,so the exploitation of MFAses with industrial value is significant for the development of maltooligosaccharides industry.This study was fouced on the MFAse derived from Bacillus stearothermophilus STB04（Bst-MFAse）.Firstly,the B.subtilis secretion expression system of Bst-MFAse was established,and the shaking flask fermentation conditions for the genetic engineering bacteria were optimized.The optimum medium was determined as:yeast extract,30 g/L;corn starch,6 g/L;KH₂PO₄,2.3 g/L,K₂HPO₄,12.5 g/L;pH 7.5.After shaking flask cultivation（200 r/min）at the the optimal temperature,37oC,for 48 h,the enzymatic activity in supernate was up to451.1 U/m L,which was 127.5%higher than the level under the initial culture condition,and10.9 times higher than the level reported.Next,the crude enzyme was purified by hydrophobic chromatography,and enzymatic properties were determined.Results showed that the optimal reaction temperature of Bst-MFAse was 75oC;its half-lives at 60,65,70 and 75oC were 120,51,24 and 13 min,respectively,and less than 5 min at 80oC.Its optimal reaction pH was 5.5,but it was most stable at pH 8.5（Gly-NaOH buffer）.Its dynamics properties were measured using different substrates.Results showed that its affinity and catalytic efficiency towards maltodextrin were the highest;both of its affinity and catalytic efficiency towards amylopectin were higher than those towards amylose.This may be due to the strong interaction between amylose chains,leading to the relatively difficult combination of Bst-MFAse and substrate,while the cluster structure of amylopectin greatly increased the probability of this combination.Besides,Bst-MFAse was dependent on metal ions;0.5 mM K⁺,Na⁺,Ca²⁺,Ba²⁺enhanced the activity slightly;while 0.5 mM Fe³⁺,Mn²⁺,Cu²⁺,Zn²⁺,Ni²⁺significantly inhibited its activity;0.5mM Na⁺or Ca²⁺could enhance its thermostability.Further study found that Na⁺and Ca²⁺improved the Bst-MFAse thermostability synergistically.The best enhancement was observed by adding 10 mM Ca²⁺and 40 mM Na⁺:the residual activity present after incubation at 80°C for 3 h（71.1%）was 4.7 fold of that when adding 10 mM Ca²⁺alone and 5.8 fold of that when adding 40 mM Na⁺alone.The crystal structure of Bst-MFAse was simulated by SWISS-MODEL,revealing a Ca²⁺-Na⁺-Ca²⁺binding site in the extension of the core,the（α/β）₈-barrel module,and it may relate to the thermostability.Results of circular dichroism spectra and intrinsic fluorescence showed that simultaneous addition of Ca²⁺and Na⁺caused the minimum change ofα-helix toβ-sheet content ratio（α/β）（19.9%）,and the least loss in maximum fluorescence intensity of protein(FI_max)（6.3%）.It was indicated that the Ca²⁺-Na⁺-Ca²⁺traid helped preserve the correct and ordered folding of the secondary structure,and stabilize the tertiary structure of Bst-MFAse.Finally,the action pattern of Bst-MFAse and the laws of its product synthesis were investigated.It was showed that Bst-MFAse could effectively degrade long-chain maltooligosaccharides（DP>6）and hydrolyze G6 after overreaction,but it was inactive toward G5.By comparison with Taka amylase A andβ-amylase,the action pattern of Bst-MFAse on starch was revealed to be endo-type.HPAEC-PAD analyses showed that both amylose and amylopectin were hydrolyzed by Bst-MFA into G1^G7,and the conversion rate of amylose was higher than that of amylopectin;but the relative proportion of main product（G5+G6）in G1^G7 in amylopectin reaction system was 24.2%higher than that in amylose reaction system.The reaction system for maltooligosaccharides production was further optimized as follows:the substrate was prepared with 5%（w/w）corn starch solution（pH 6.0）,and then hydrolyzed by 5 U/g of Bst-MFAse at 60°C for 48 h.The conversion rate of substrate and main product ratio reached 74.6%and 58.8%,respecively.The conversion rate was further increased to 101.2%by simultaneously adding 2 U/g of pullulanase in the beginning of the reaction,while the main product ratio still remained 55.0%.