Secretory Expression Of The Maltopentaose-producing α-amylase Gene And Maltopentaose Preparation Technology

α-amylase is classified as an endoamylase which hydrolyses α-1,4 glycosidic linkages in starch.Starch is therefore broken down rapidly to such as glucose,maltose and malt-oligosaccharides.The components of products obtained by hydrolyzing starch with different α-amylase vary greatly.Some α-amylase hydrolyzed starch products have the highest glucose content,some have the highest maltose content,and some have the highest malto-oligosaccharide content.The results show that malt-oligosaccharides have good nutritional and functional properties.However,it is difficult to prepare single maltoligosaccharides with high purity,the higher degree of polymerization the more difficult,like maltopentaose and maltohexaose.In addition,the preparation technology has been monopolized by a minority of countries for a long time,which greatly limits the further research and application of malt-oligosaccharides.In the preliminary work,a B.subtilis strain capable of secreting high-yield maltopentaose α-amylase(HA1)was screened,and the amylase gene ha1 was cloned.However,ha1 is expressed in E.coli with a certain amount of inclusion bodies,and the food safety of the E.coli expression system also limits the range of applications of its products.Therefore,this paper intends to introduce ha1 into B.subtilis strain WB600 for secretory expression and study the preparation process of maltopentose,in order to solve the food safety problem of E.coli expression system and simplify the preparation of amylase.The main results are as follows:(1)The expression vector of B.subtilis with ha1 gene was constructed,and the enzymatic properties of α-amylase(HA1B)showed that its optimal p H and temperature were 6.5 and 45°C,and it had better stability at p H 7.5 and 30°C.The ha1 gene recombinant bacteria were cultured in 250 m L shake flasks in 50 m L medium,and the extracellular crude enzyme activity could reach 198 U/m L.In addition,analysis of the starch hydrolysate of recombinase HA1 B showed that the hydrolysates of B.subtilis recombinase(HA1B)and E.coli recombinase(HA1E)were different: The largest content of HA1 E hydrolyzed starch products is maltopentaose(about 65%).By controlling the time of hydrolyzing starch and the amount of enzyme added,the largest proportion of HA1 B hydrolyzed starch products is maltopentaose(about 60%)or maltose(about 75%).(2)Optimize the preparation,separation and purification conditions of maltopentose,including nine aspects: the reaction temperature and p H of hydrolyzed starch,the amount of enzyme added,the reaction time,the type of purified substrate,flow rate,temperature,sample volume,sample concentration and eluent types.The results showed that the best conditions for HA1 E to hydrolyze starch to produce maltopentose was: at 30°C,p H 6.5,and enzyme amount of 16 U/m L,react with 2% corn starch for 8 h,and the maltose content was 66%.The best separation and purification conditions of maltopentaose are:using macroporous resin AB-8,eluting with deionized water at a flow rate of 0.7m L/min under the conditions of 65°C,1 m L loading volume,and 486.64 mg/m L loading concentration.Maltopentaose with a content of over 93% and a recovery rate of about 74%.