Heterologous Expression Of Maltopentaose-forming Amylase And Analysis Of Its Enzymatic Properties And Product Synthesis

Maltooligosaccharides,composed of 3 to 10 ?-D-glucopyranoses linked by ?-1,4-glycosidic bonds,are novel functional oligosaccharides which are promising in food industry for the favorable processing adaptability and unique physiological function.Among them,maltopentaose(G5)is highly valued in medical and pharmaceutical fields due to its utility in diagnostic reagent for the detection of ?-amylase in serum and urine.At present,the market for maltopentaose is monopolized by a few developed countries such as the United States and Japan,leading to high monopoly price.Therefore,it is imperative to develop high-valued maltopentaose with independent intellectual property rights.The preparation of maltopentaose mainly uses maltopentaose-forming amylases(G5-amylases),which has the ability to hydrolyze starch.However,G5-amylases reported so far have problems such as low catalytic activity and poor thermostability,and cannot meet the requirements of industrial applications.Consequently,it is necessary to obtain gene of G5-amylases with excellent potential value through gene mining methods,and carry out heterologous expression in genetically engineered bacteria to improve the fermentation intensity.In addition,the products of G5-amylases hydrolysing starch are usually maltooligosaccharides mixture,which causes production costs in subsequent separation and purification of G5.Thus,it is of great significance to study the regulation strategy for the directed product synthesis of G5-amylase with the purpose of promoting efficient production of G5.This study screened the gene of G5-amylase(BmMFAse)derived from Bacillus megaterium STB10,and the Escherichia coli BL21(DE3)and Bacillus subtilis WB600 expression system were established,with the secretory expressions of BmMFAse being proceeded.Results showed that B.subtilis WB600 was the optimal expression system for BmMFAse,and the shaking flask fermentation conditions for the genetic engineering bacteria were optimized.The optimum fermentation conditions were determined as: yeast extract,36 g/L;potato starch 7 g/L;KH2PO4,2.3 g/L,K2HPO4,12.5 g/L;pH 7.0.After shaking flask cultivation(200 r/min)for 60 h at the optimal temperature,25?,the catalytic activity in fermentation supernatant was 2.21 times higher than that under the initial fermentation conditions.Secondly,the enzymatic properties of BmMFAse were studied.Results showed that the optimal reaction temperature of BmMFAse was 50? and the optimal reaction pH was 7.0,and BmMFAse exhibited excellent thermostability.When BmMFAse acted on soluble starch,maltodextrin and raw starch,the affinity and catalytic sufficiency of the enzyme to soluble starch were highest among other substrates.In addition,the affinity of BmMFAse to amylopectin was much higher than that of amylose,the dynamics properties laid the foundation for the selection of suitable substrate for BmMFAse.In addition,the action pattern of BmMFAsewas indicated to be endo-type.Furthermore,BmMFAse could produce G5 in absolute predominance on the degradation of maltooligosaccharides with DP>6,substrates with high amylopectin content were beneficial to the formation of the main product G5 and the progress of the reaction.Whereafter,the fusion protein strategy was used to improve the product specificity of BmMFAse.It is found that fusing CBM20 at the C-terminal of BmMFAse to construct a fusion enzyme,namely,BsMFAse,brings about increase in the hydrolysis activity and product specificity.The result of product synthesis of MFAses were proved using HPAEC-PAD.The results showed that the fusion protein hydrolyzed the intermediate products G6 and G7 to a greater degree,thereby increasing the proportion of the main product G5.Furthermore,the increased G5 ratios when BsMFAse hydrolyzing tapioca starch,maltodextrin,corn starch and soluble starch were respectively 38.30%,10.23%,14.99% and26.32% when compared with BmMFAse.Finally,the industrial application of BsMFAse was simulated.we explored preliminarily the ability of BsMFAse to produce G5 with 20%(w/w)topicia starch as the substrate.The substrate slurry was preheating at 60? for 5 min and liquefied at 95? for 15 min.The conversion rate of substrate and main product ratio reached 92.67% and 47.71%,respectively,by incubating 25 U/g of BsMFAse and 5 U/g of pullulanase with substrate slurry at 40?.