| Beta-amylase(EC 3.2.1.2),also known as maltosidase and glycogen amylase.It is widely used in the industrial field such as brewing,food processing and medicine.Expecially,?-amylase and other starch hydrolases are complexed to produce high purity maltose syrup.?-amylase is first found in higher plants.Compared with plant ?-amylase,the microbial ?-amylase has the advantages of high purity,little limitation with the supply of raw materials,and the suitability for large-scale production.In this study,?-amylase from Bacillus flexus CCTCC2015368 was expressed in Brevibacillus choshinensis,then optimized by shake flask and 3-L tank fermentation to improve the yield of the recombinant ?-amylase.The recombinant ?-amylase was purified by ammonium sulfate precipitation and size exclusion chromatography to investigate the enzymatic properties.After this,the ability of the recombinant ?-amylase convert starch into maltose was studied.To decrease the optimal pH of the recombinant ?-amylase,the site-directed mutagenesis was performed and the ability of mutants to convert starch into maltose was studied.The main findings of this paper are as follows:(1)In this study,the ?-amylase gene from Bacillus flexus CCTCC2015368 was expressed in Brevibacillus choshinensis.The recombinant strain B.choshinensis/ pNCMO2-amy was cultured in TM medium,and the enzyme activity was 1669 U?mL-1after 48 h fermentation.SDS-PAGE analysis showed one major protein band corresponding to a protein of approximately 57.6 kDa,indicating that the ?-amylase gene derived from B.flexus was successfully expressed in B.choshinensis.(2)The fermentation conditions of B.choshinensis/pNCMO2-amy were first optimized at the level of shake flask.The results showed that 15 g?L-1 soybean peptone and 5 g?L-1 beef extract were used as nitrogen source,and the initial carbon source was 20 g?L-1 Glucose,the amount of enzyme production reached the maximum level of 2516 U?mL-1.Then the concentration of nitrogen and the fermentation temperature were further optimized at the level of 3-L fermentor.The results showed that the highest enzyme activity was achieved when the concentration of nitrogen was 20 g?L-1 and 40 h at 35? and the ?-amylase activity in the culture supernatant reached 4472 U?mL-1.(3)The recombinant ?-amylase was purified by ammonium sulfate precipitation and size exclusion chromatography to investigate the enzymatic properties.The recombinant ?-amylase had a specific activity of 2656 U?mg-1using soluble starch as the substrate.Its optimal temperature and pH were 50? and 7.0,respectively,and its half-life when incubated at 50? and pH 7.0 was 34 h.The kinetic constants(Km,Vmax and kcat)of the recombinant ?-amylase were 3164 U?mg-1,1.84 mg?mL-1 and 2373.3 s-1,respectively.(4)The production of maltose using recombinant B.flexus ?-amylase with 30%(w/v)potato starch as substrate was also optimized.At pH 6.5 and 55?,when the amount of ?-amylase was 200 U?g-1dry starch,then mixed with isoamylase and maltose amylase,the yield of maltose reached 86.9%.These results suggest that this recombinant B.flexus ?-amylase is suitable for use in the industrial production of maltose.(5)However,the optimum pH of the recombinant ?-amylase is 7.0,which makes it difficult to complex with other starch hydrolases.In the present study,three B.flexus ?-amylse mutants T47 K,Y164K,L396 K were constructed and the optimal pH were changed from 7.0 to 6.0,4.5 and 5.5 respectively.At the same time,the optimum temperature of L396 K was increased from 50? to 60?.The production conditions of maltose using L396 K ?-amylase was also optimized.When using potato starch(10% w/v)as the substrate under the condition of pH5.5 and temperature 60?,then mixed with 8 U?g-1 pullulanase and15 U?g-1 maltogenase,the yield of maltose reached 91.3%,which satisfied the production requirement of ultra-high purity maltose syrup. |
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