Site-directed Mutagenesis Of Thermostable ?-amylase And Its Expression In B.subtilis

Amylase is one of the most important industrial enzyme which takes up more than 50% of the total market of enzyme production.Among them,thermostable?-amylase has gained more attention and has been widely used in food,fermentation,textile,and paper industry due to its high thermostability.At present,most of the industrial thermostable ?-amylase is Bacillus licheniformis ?-amylase,namely BLA.However,the natural BLA production capability of Bacillus licheniformis is relatively low and can not meet the great need of BLA's industrial application.Therefore,developing an efficient heterogenous expression system for industrial production of BLA has become an priority of amylase researches.Meanwhile,searching for new thermostable ?-amylase with more desirable properties than wild BLA,has also become the main task for researchers.In this study,gene amyLM,which codes for a mutant BLA with two amino acid mutations,N190 F and Q264 S,was designed and synthesized.the mutation sites were decided based on the recommendation in patent that these mutations potentially make the protein has higher thmostability and less Ca2+ independence.Then amyLM was successfully expressed in B.Subtilis 168,enzyme activity of amyLM in fermentation supernatant is 46U/ml.In order to improve the expression efficiency of amyLM,the expression host was changed from B.Subtilis 168 to B.Subtilis WB800 n,and the usage of promoter P43 was optimized on sequence level.Three expression vector with different sequence distance of 9bp,18 bp,35bp between amyLM and the SD sequence of P43 were constructed,the results showed that when amyLM was 9bp downstream the P43 SD sequence,the supernant of WB800 n fermentation achieved the highest BLA enzyme activity of 422U/ml,which is 9.17 times of that when amyLM was first expressed in B.Subtilis 168.The results has shed some light on how to make the most effective use of P43 when it comes to foreign protein expression in B.Subtilis.Also in this study,both BLA wild gene amyL and mutant gene amyLM were expressed in WB800 n using maltose inducible promoter Pglv,and enzyme activity in supernatant of amyLM was 268U/ml,which was 1.86 times of that in supernatant of amyL,which was 144U/ml.