Heterologous Expression And Thermostability Modification Of Bacillus Pullulanases And Their Application In Vermicelli Production

Vermicelli is a common starchy food.Among various forms of vermicelli,potato vermicelli is favored by many consumers because of its rich nutrition,low calories,and high vitamin and lysine contents.However,raw potato materials have a high amylopectin content and are not easily aged.Fortunately,the degree of aging can be enhanced by adding a certain amount of alum so that the produced vermicelli is tough and does not easily break.However,excessive alum intake causes excessive aluminum contents in the human body and endangers human health.In a previous research,vermicelli production is developed by using pullulanase instead of alum to obtain high-quality potato vermicelli.In the present study,the pullulanase gene derived from Bacillus was expressed in different hosts.The enzymatic properties,thermostability modification,and application of recombinase in vermicelli production were explored.The main contributions of this study were as follows:（1）The pullulanase-encoding gene Bs P of Bacillus subtilis 168 strain was cloned and expressed in Escherichia coli JM109,B.subtilis WB600Δamy E,and B.subtilis 1A717.In E.coli,the recombinase Bs P accumulates intracellularly.In B.subtilis WB600Δamy E and1A717,Bs P is secreted outside the cells.The specific activity of the recombinant enzyme Bs Bs P expressed in B.subtilis and secreted to the culture medium was 153.7 U·m L^-1,which is 4.5-fold greater than that of the products accumulated in the cytoplasm of E.coli.The optimum temperature and p H of Bs Bs P is 45℃and 6.0,respectively.And it has good stability in the range of p H 5.0 to 7.0.Its K_m and V_max are 2.20 mg·m L^-1 and 0.769μmol·m L^-1·min^-1,respectively.Bs Bs P degrades pullulan to produce a single product,namely,maltotriose,which has no degrading effect on amylose.It is a type I pullulanase that can be used for vermicelli production.（2）The pullulanase-encoding gene Gs P6837 derived from Geobacillus sp.XQ6837 was cloned and expressed,and the recombinase Gs P6837 was secreted to the periplasm and extracellular environment of E.coli JM109 to varying degrees.Its extracellular,periplasmic,and intracellular enzyme activities are 198.18,109.65,and 248.27 U·m L^-1,respectively.The extracellular enzyme activities of Gs P6837 in B.subtilis WB600Δamy E and 1A717 reach269.48 and 317.19 U·m L^-1,respectively.The specific enzyme activity,optimum temperature,and optimum p H of Gs P6837 are 202.45 U·mg^-1,65℃,and 6.5,respectively.Its half-lives at65℃and 70℃are 4.01 h and 7.46 min,respectively.The activity of Gs P6837 can be promoted by Ca²⁺,Mg²⁺,K⁺,Na⁺,and Co²⁺but can be inhibited by Cu²⁺,Zn²⁺,Mn²⁺,and Fe²⁺.Its k_catand K_m are 260.76 s^-1 and 5.21 mg·m L^-1,respectively.Gs P6837 is a type I pullulanase,which is suitable for vermicelli production.（3）The thermal stability of Gs P6837 was modified on the basis of a semi-rational design,and H255G and the combined mutation H255G/S351V/Q87F/Q459K were obtained.H255G and H255G/S351V/Q87F/Q459K have T_m of 77.54℃and 78.29℃,respectively.T_m increases by 2.34℃and 3.09℃,respectively.The half-lives of H255G,and H255G/S351V/Q87F/Q459K at 70℃are 22.58 and 23.75 min,respectively.The half-lives of H255G and H255G/S351V/Q87F/Q459K at 70℃are 3.02 and 3.18 times that of wild-type Gs P6837.The specific enzyme activity and K_m of the combined mutation are 222.58 U·mg^-1 and 4.50,respectively,and these values are better than those of wild-type Gs P6837.The optimal temperature of Gs P6837,H255G,and H255G/S351V/Q87F/Q459K is 65℃.Circular dichroism analysis shows that the secondary structures of H255G,H255G/S351V/Q87F/Q459K,and wild-type Gs P6837 are similar.（4）The fermentation medium was optimized at the shake flask level.The optimal carbon source was 20 g·L^-1 sucrose,the optimal nitrogen source was 25.4 g·L^-1 soybean cake powder,and the inorganic salt source was 0.99 g·L^-1 Na Cl.After optimization,the enzyme activity of Gs P6837CM is 439.05 U·m L^-1,which is 1.76 times the initial value.And the enzyme activity of Bs Bs P is 35.28 U·m L^-1 after optimization,which is 1.5 times the initial value.The recombinant strain was cultured in a 5 L fermentor with the optimized medium in the shake flask,and the highest enzyme activity of Gs P6837CM was 705.75 U·m L^-1,which was 1.61times the level of the shake flask.Therefore,Bs P,Gs P6837 and Gs P6837CM can be used in enzymatic vermicelli production.Among them,Gs P6837CM can produce potato vermicelli with a good quality under the enzymatic reaction conditions at 70℃.