Genetic Improvement And Fermentation Process Optimization Of α-amylase Producer

The thermostableα-amylase is one of the most important commercial enzymes with a number of applications, including its use in starch processing, sugar, textile and paper industries. Up to the present, Bacillus licheniformis is the only industrial strain for production of thermostableα-amylase. This study has investigated the genetic improvement and fermentation process optimizing of the industrial strain Bacillus licheniformis, hoping for increasing the expression level of the thermostable a-amylase.Based on the optimization the protein expression system of the thermostableα-amylase producing strain B. licheniformis, the aim of the present work is to perform the genetic improvement of B. licheniformis by genetic engineering. In this work, a 600 bp full gene fragment of degQ that amplified from the genomic DNA of B. licheniformis by PCR method, was digested with BamHI and inserted into the BglII site of pHY-P43, produced a recombinant plasmid pHY-P43-degQ. The recombinant plasmid pHY-P43-degQ was transformed into B. licheniformis ATCC14580 and B0204 by electroporation method, resulted in recombinant strains ATCC14580 (pHY-P43-degQ) and (pHY-P43-degQ), respectively. The results of shaking flask fermentation indicated that theα-amylase production level by strain ATCC14580 (pHY-P43-degQ) was 35.4 U/ml, which is almost 30 folds of the original strain (1.2 U/ml), and that of strain B0204 (pHY-P43-degQ) increased 15-18%. A further study has done to investigate and optimize the fermentation process of the recombinant strain B0204 (pHY-P43-degQ).The studies on the physiological characters of strain B0204 (pHY-P43-degQ) showed that it was able to utilize glucose, lactose, sucrose, glycerol, galactitol, galactose, sodium gluconate and gluconate, and can grow at a temperature range of 34~50oC and at a pH range of 5.5~7.8 with its optimal growth conditions of temperature of 42 oC and pH of 6.5, respectively.This study has initially confirmed the proper medium and fermentation process, and the optimized shaking flask fermentation conditions were:bean power 2.5 %,lactose 4 %, PO4-3 40 mmol/L, pH 6.0, inoculation 10%. After the optimization of the fermentation, the thermostableα-amylase produced by the recombinant strain B0204 (pHY-P43-degQ) was increased 71.5 %. On the optimized fermentation condition, the recombinant strain B0204 (pHY-P43-degQ) produced 28 % thermostableα-amylase more than that of strain B0204.