Fermentation Process Optimization Of Thermostable ?-amylase Producer BL-H19

Thermostable a-amylase is one of the most important commercial enzymes with various applications including sugaring,fermentation,brewage and textile industries owing to its thermal stability.This study has investigated the fermentation process optimization of thermostable a-amylase of Bacillus Licheniformis,hoping for increasing the expression level of the thermostable a-amylase.Strain BL-H19 was identified by morphology,physiology and biochemistry analysis,and its optimal culture condition of shaking flask seed was identified.Growth curve and amylase production curve of strain BL-H19 was drawn,and a-amylase production is lag type.The optimum temperature of thermostable a-amylase is 86 ? and the optimum pH is 7.0.The fermentation condition and medium had been studied by single factor method and response surface methodology.The fermentation medium consists of soybean powder 2.6%,cottonseed powder 2.1%,(NH4)2SO4 0.5%,liquefacient starch 12.0%(DE is 24 ± 1),liquefacient starch 5.3%,CSL 3.0%,CaCl2 0.045%,K2HP04 1.4%,KH2P040.6%,sodium citrate 0.2%,pH value about 6.5.The fermentation condition is inoculating volume 4%,fermentation volume 50mL/250mL stirring rate 260r/min and temperature 40?.In these optimum conditions,the thermostable a-amylase activity was 4393u/mL in 7 days,which was 2.02 times before optimized.By the study of culture technology and feed technology in 10L fermenter,the optimum fermentation process was identified.The seed was inoculum enlarged after 24 h during two steps fermentation and the inoculating volume is 10%.The fermentation process is divided into two stages of growth and fermentation.From 0 h to 48 h,culture temperature was 38?,pH value was kept at about 6.5 by automatic feeding ammonia,DO value was kept at 30-35,the foam was under control.From 48 h to 102 h,culture temperature was 40?,DO value was kept at 30-50.From 60 h to 96 h,liquefacient starch was feeding as flow rate 7.5mL per minute to maintain the fermentation by controlling the concentration of total sugar between 1.6%and 1.9%,the concentration of reducing sugar between 0.9%and 1.3%,stability of pH.In these optimum processes,the thermostable a-amylase activity was 10373u/mL in 102 h,which was 2.07 times of the previous,the output was 2.35 times of the previous and the fermentation period was shortened by 15%.