Discovery,Characterization And Thermal Stability Mechanism Of A Novel Acid Resistant Thermal Stable ?-amylase

Alpha amylases,which are the first commercial enzymes and used most widely,and have the largest dosage,are indispensable in starch sugar and fermentation industry.The optimum pH of commercialization acid and thermal-stable alpha amylases which are widely used in industry is 5.0-6.0.These amylases easily inactivating at high temperature of deep processing of starch liquefaction process.In addition,the optimum pH of glucoamylases used in subsequent saccharification process is 4.5.The pH adjustment between the two steps will increase the cost.So the development of new type acid and thermal-stable alpha amylases which liquefy starch under low pH value(4.5)efficiently is significant for industrial application.This study is around the molecular modification of gene of acid and thermal-stable alpha amylase from Bacillus licheniformis.The structural biology and computer simulation technology are used to rationally design molecular modification,the traditional biochemical technologies are used to determine the enzyme properties and thermal stability.This study constructs 5 mutants from known literatures,acid and thermal-stable alpha amylases from Bacillus licheniformis and commercial acid and thermal-stable alpha amylase.These seven alpha amylases are expressed and purified at same conditions.The results of the enzymatic properties analysis of the seven kinds of alpha amylases are listed below:the thermal stability of mutants BLA-m6 and BLA/m9 under the condition of pH4.5 is obviously better than and other mutants and BLA.The mutant BLA-m9 maintains high specific activity at pH4.5-5.0 range,which indicates its high acid resistance.In the following comparison of sequence,it is found that the sites M15T and N188S may have positive effects on thermal stability.On the basis of the above study results,BLA-m9 is used to be template to build a single site mutants include of M15T,N188F,N188P and N188S and combine mutants M15T/N188F,M15T/N188P and M15T/N188S.The expression and purification,enzymology properties analysis and thermal stability analysis of these seven mutants based on BLA-m9 are processed.At the same time,the spectroscopy technology and dynamics simulation technology are used to analyze the structure of alpha amylase,and the thermal stability mechanism with changes of Met15 and Asn188 sites is researched on the molecular level.It is found that BLA m9-M15T,BLA m9-N188S and BLA-m9-M15T/N188S maintaining the initial activity of 100%,91%and 91.5%,respectively,and are all significantly higher than the 60%of BLA-m9.And the specific activity within the scope of the pH4.0-5.0 increased significantly compared with BLA-m9.As for BLA m9-M15T,its thermal stability under acid condition improved and its expression increased at the same time compared with BLA-m9.BLA-m9-M15T has a good prospect in industrial application as a result.The analysis of fluorescence spectrum shows that the red shift phenomenon of these three mutants above is not obvious with the temperature increase from 25 to 95 ? its fluorescence spectrum,which indicating its tertiary structure can keep high integrity at high temperature.The secondary structure content is calculated according to the circular dichroism spectra.It is found that secondary structure remains relatively complete at high temperature.The structure simulation results show that the hydrogen bond network of the 15 site of mutants BLA-m9-M15T and BLA-m9-M15T/N188S and the 188 site of BLA m9-N188S and BLA-m9-M15T/N188S mutants show enhancement compared with BLA-m9.The results of dynamics simulation reflect these three mutants are with higher flexibility than BLA-m9.All of these results indicate Met15 and Asn188 are the key residues to maintain BLA thermal stability under acid condition.And the mechanism of improvement of thermal stability with M15T and N188S mutations is revealed in molecular level.This method of structure prediction based on the reasonable design has important guiding significance for other industrial enzymes to enhance the thermal stability.The research method of connecting protein structure with function used spectral technology and simulation technology for proteomics research is of great significance.