Heterologous Expression And Thermostability Engineering Of Bacillus Stearothermophilus α-amylase

Alpha-amylases,which hydrolyze theα-1,4-glucosidic linkages of starch or related carbohydrates into dextrin,oligosaccharide,maltose and glucose,are used extensively in pharmaceutical,textile,food,and detergent industries.Among the variousα-amylases,Bacillus licheniformisα-amylase（BLA）is widely used in industrial starch hydrolysis processes for its remarkably thermostability.However,BLA needs Ca²⁺to maintain its stability and activity.The maltohexaose-formingα-amylase（AmyMH）secreted by the strain Bacillus stearothermophilus WSH13-17,which was isolated in our laboratory previously,has a remarkable catalytic efficiency and Ca²⁺independence.The catalytic efficiency of AmyMH is about 8-fold to that of BLA.Hovever,the thermostability of theα-amylase could not match application requirement.In the present study,the Bacillus stearothermophilusα-amylase was expressed in Escherich coli and Brevibacillus choshinensis.To improve the production of AmyMH,fermentation conditions and culture medium were optimized.In addition,the thermostability of AmyMH was improved through tructure-based rational design.After fusion with oligopeptides at N-terminal domain of AmyMH,the thermostability of AmyMH could be further improved.The main results are listed below:（1）Heterologous expression and characterization of AmyMH in E.coli were investigated.Based on the preferrd codons of E.coli,the codons of AmyMH was expressed in E.coli BL21（DE3）.Optimization of single peptide for the extracellular expression of AmyMH in E.coli BL21（DE3）was initiated.The result showed that the extracellular production of AmyMH could be greatly improved through replacement of the native signal peptide(173 U·m L^-1)by PelB signal peptide(782 U·mL^-1).However,the fermentation result showed that a higher production of AmyMH is always combine with a fewer cell concentration.Furthermore,the TEM-image of recombinant E.coli showed that most of cells are destroyed.In addition,when the inactivated AmyMH was expressed in E.coli BL21（DE3）,most of cells displayed normal state.Thus,the expression of AmyMH was toxic to E.coli BL21（DE3）.The AmyMH was also expressed in E.coli C41（DE3）,which is a mutant strain from E.coli BL21（DE3）.Theα-amylase expressed in E.coli C41（DE3）could reach 1560 U·m L^-1,which was 2-fold that of expressed in E.coli BL21（DE3）.The optimum temperature and pH of AmyMH was 70℃and 5.5.The specific activity,k_cat,K_m and k_cat/K_m was 5.3×10⁴ U·mg^-1,6.5×10⁴ s^-1,2.1 g·L^-11 and 3.1×10⁴ g·L^-1·s^-1,respectively.The half-life of AmyMH at 95℃,pH 6.0 and 1 mM Ca²⁺was 60 min.When the temperature increased to 110℃,the enzyme was quickly inactivated.（2）The AmyMH gene was expressed in Brevibacillus choshinensis and the fermentation conditions and culture medium were optimized to improve the production of recombinant protein.The AmyMH gene from B.stearothermophilus was inserted into the expression vector pNCMO2,and then expressed in B.choshinensis SP3 in shake-flask.Theα-amylase activity produced by recombinant B.choshinensis SP3 could reach 2149 U·m L^-1,which was 1.4-fold that of produced by E.coli.The optimization of culture medium and fermentation conditions resulted in an 8-fold improvement inα-amylase yield,which reached 1.72×10⁴ U·mL^-1.Addition of proline to the culture medium significantly improved the production of recombinantα-amylase in B.choshinensis SP3.This improvement may result from improved cellular integrity of recombinant B.choshinensis SP3in existence of proline.And this improvement might be a universal phenomenon.（3）The effect of un-conservative asparagine in domain B on thermostability of AmyMH was investigated.Domain B is crucial for thermostability ofα-amylase and deamidation of Asn emerges as the cause of inactivation.By using multiple sequence alignment,three un-conservative asparagine（Asn122,Asn149 and Asn193）were selected for mutation.Among five mutants（N122D,N149S,N149D,N193H and N193F）in this study,the thermostability of N193F mutant displayed the largest improvement in thermostability,which was 2-fold that of the wild-type enzyme at 95℃,pH 6.0 and 1 mM Ca²⁺.The structural model showed that Asn193 is located in a long Loop around Ca²⁺-Na⁺-Ca²⁺binding site.This long loop might play a key role in determining the overall stability of AmyMH.（4）The thermostability of AmyMH was improved through immobilized a long loop（178-200）in domain B.Through comparison of a homologous model structure of AmyMH with the crystal structure of the thermostableα-amylase from Bacillus licheniformis,four sites（Pro245,Ser242,Gly180 and Ile181）were chosen for mutation.Five mutants carrying the mutationsΔIG,S242A,ΔIG/N193F,ΔIG/N193F/P245R andΔIG/N193F/S242A were generated and their proteins characterized.EDTA inhibited the activities of the wild-type enzyme and its mutants.AndΔIG/N193F/S242A was the best EDTA-resistant mutant.In addition,ΔIG/N193F/S242A displayed the highest activity（60%）after incubating at 60℃in the presence of 10 mM EDTA.Thus,the Ca²⁺binding inΔIG/N193F/S242A was the tightest.TheΔIG,S242A,ΔIG/N193F andΔIG/N193F/S242A mutants displayed longer half-life.The most thermostable mutant protein,ΔIG/N193F/S242A,exhibited a 26-fold and 5-fold improvement in half-life at 95℃in the absence and presence of added Ca²⁺,respectively.The effect of immobilized corresponding loop in domain B on thermostabililty of BLA was also investigated.The most thermostable mutant,A269K/S187D/N188T,exhibited a half-life of 270 min,which was 9-fold improvement at 95℃and pH 5.5,compared with that of the wild-type enzyme.（5）After fusion with four different oligopeptides at N-terminal ofΔIG/N193F/S242A mutant,recombinant enzymes were expressed and characterized in E.coli.Theα-amylase activities expressed in E.coli after fusion with oligopeptides were similar with that of control.OP1-ΔIG/N193F/S242A displayed a better EDTA resistant performance,and another three mutants did not enhance EDTA resistant performance compare to that before fusion.The thermostability of OP1-ΔIG/N193F/S242A was improved as well.After incubated at 95℃,pH 6.0 and 1 mM Ca²⁺for8 h,the activities of OP1-ΔIG/N193F/S242A andΔIG/N193F/S242A remained 58%and 37%,respectively.The thermostability of OP1-ΔIG/N193F/S242A was also compared with commercialα-amylase at 110℃,the result showed that the thermostability of OP1-ΔIG/N193F/S242A was better than that of commercialα-amylase at low comcentration of Ca²⁺（1 and 5 m M）.