| In laboratory condition, above all a preliminary study was done regarding the strain growing and zymogenic metabolism of a genetic engineering strain TR12 of Aspergillus Kawachii which contains fusion genes of multi-copy α-amylase and saccharifying enzyme by shaker-flask culture. Through investigation of many factors such as medium composition, fermentation cycle, medium volume, initial pH value, seeding volume, etc., and through optimization of these factors, a new liquid phase fermentation was found through primary fermentation process to obtain two sorts of new enzymic preparations containing two high enzyme activity. Also through fusion breeding technology of natural separation and purification culture and protoplast, the practical strain has obtained genetic characteristics of high growing speed and stable activity for enzymic production.Through further study on the feed-batching quantity, time, batches and culture medium of genetic engineering strain TR12 of Aspergillus Kawachii in liquid phase, an optimum feed-batching process for zymogenic metabolism. In this process, the volume of fermenting liquor increased bit by bit after three feed batches. Total enzyme activity produced by TRI2 is steadily increased. After 96 hours of fermentation, the activity of acid-resistant a-amylase and saccharifying enzyme reached a high level of 86.37u/mL and 25229.7 u/mL respectively. The result of 2L small fermentation drum also proved that shaker-flask fermentation level can be reached under scale-up air-agitating fermentation conditions.The fermented liquor was extracted and separated to obtain about 70% yield with a novel enzyme preparation with liquefying and saccharifying enzyme activity. Its optimum pH value was 4.6 and the optimum temperature of the acid-resistant a-amylase and glucoamylase was 60 ℃ and 50 ℃ respectively. At pH4.6, the enzyme activity was stable at 50℃ for 1 h and its activity was enhanced by Ca2+, while inhibited by Fe3+, A13+, etc.Through investigation of mixing ratio, culture temperature, humidity, culturing time and ventilated air quantity of wheat bran medium in solid brewery fermentation, an optimum fermentation process was found to prepare a novel type of moldy bran crude enzymic preparation. The activities of its acid-resistant a-amylase and saccharifying enzyme respectively reached a high level of 86.64 u/g and 54 147.4 u/g. Activity of acid-resistant a-amylase is dozens higher than the common bran-qu used in current breweries. The result of liquor fermentation shows that residual starch amount dropped about 6%. Starch transglucosylation is high and alcohol yield is high. Alcohol quality has no difference from conventional production.In alcohol fermentation test in laboratory, adjust starch slurry pH to 4.6, no paste, add self-made enzymic solution 10 u/g starch (as acid-resistant a-amylase) at 60℃ liquid saccharifying for 1 h, temperature dropped to 50℃, made up self-made enzymic solution 80 u/g starch (as acid-resistant a-amylase) to saccharify 30 minutes, used for alcohol fermentation, alcohol yield is 49.44 ml/100 g starch. The test reached a high level of theoretical yield of 94.3%.In lab preparation of starch sugar and preserved fruit syrup, use 30-32% starch slurry to adjust pH 4.5-4.6, no paste, add self-made 10 u/g dry feed (as acid-resistant a-amylase) at 60-65 ℃ temperature to liquefy for about 60 minutes, with temperature dropped to 55℃, add self-made 100 u/g dry feed (as acid-resistant a-amylase) to continue saccharification for 20-25 h. The saccharifying DE value reached an ideal level of 96%-98%.The successful research of the efficiency acid-resistant saccharifying enzyme preparation not only makes a record for enzymic preparation varieties inenzymic preparation industry in China, but also brings a prospective application in improving the hydrolysis process of starch sugar industry, increasing raw material utilization ratio of brewery, bio-transferring of spent dross in alcohol industry as well as the development of stomach new medici...
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