Microbial Fermentation Production Of Acid Alpha-amylase

Recently, as our country's starch processing industry development so quickly, the process technology has been changed. More and more factory needs the new kinds of amylase to be suit for the new methods. Now, in the national and native markets people can only find theα-amylase is functional in high or low temperature and also the alkaline conditions, its suitable pH is from 6-10. Under the acidity conditions, its major activity will be lost, so can not do the starch processing under the acidity conditions. Then the needs of a novelα-amylase can tolerance the acidity condition are more urgent. So our research is a systemic work about how to produce the acid-stableα-amylase by the microbial fermentation. It includes: breeding the acid-stableα-amylase producing strains, enzymology characteristic research of the acid-stableα-amylase, conditions optimization of the liquid fermentation of the enzyme producing strain, isolated and purified of the acid-stableα-amylase. The results are as follows:1. The high acid-stableα-amylase productivity strain Aspergillus niger Tx-78 was isolated from mold culture. After the UV and NG mutation treatment, the mutant strain can produce the target enzyme 1120U/mL. It is higher than the originate strain 86.7%. And also this mutant has a nice genetic stability.2. By the enzymology characteristic research of the acid-stableα-amylase produced from Mutant strain UNTx78-358, the enzyme was identified as an exoenzyme. Its reaction optimum temperature and pH is 70℃and 4.0 respectively. The kinetics research shown its Km is 7.89g/L. And Ca²⁺ cans accelerant the activity of this enzyme remarkable, especially when the consistency of Ca²⁺ is 7mmol/L. The results of TLC demonstrated that the products from starch catalyzed by the enzyme were mainly maltose and glucose. The results show this enzyme has a good acid tolerant and thermo stability, which can achieve the liquefaction and saccharification of the starch spontaneously. So it will have a good application prospect.3. By the optimum fermentation medium research of UNTx78-358. The results show: the optimum culture conclude starch 6.23g/L, peptone 12g/L, CaCl20.287g/L respectively, and phytic acid 1‰. The optimum fermentation conditions are as follows: 100ml/300mL per flask, 10% inoculums concentration, 48h cultivate on 30℃. After that, replenish 20ml culture then continue cultivate until 96h, and the pH is 4.0. By this experiment, the production of the enzyme can be 2400/mL, is higher than the original production 114.3%.4. The isolated processing of the enzyme producing strain is as follows: The crude enzyme fraction can get by 8 layer gauze filter or 200 seive mesh . Adjust the pH to 6.0 of the crude enzyme fraction then concentrate it by rotary evaporator under the 0.09Mpa, 55℃. Then concentrate the enzyme fraction by 3.5 times ethanol under 4℃. Collect the precipitate and freeze drying it under -50℃, 24h. After that, the enzyme can be isolated. Its activity can remain 89.3%, purified times is 2.8, yield is 8g/L. The enzyme was purified by CM52 and DE52 cellulose ion-exchange chromatography and SDS-PAGE. The results show the mass of this enzyme is about 74kD. And its N-terminal amino acid sequence is Leu, Ser, Ala, Ala, Glu, Thr, Arg, Thr, Gln, Ser, Ile, Tyr, as same as the sequence of acid-stableα-amylase product from A. niger in other reports.The aim of this reaserch is provide the useful statistics for the development of the acid-stableα-amylase in our country by the isolation and screening high yield strain, enzymology characteristic research, optimum fermentation conditions design and isolated processing research. For that reasons, this research not only can optimize for maximum profit of our countries production of acid-stableα-amylase, But also will be taking higher position in terms of academic and economic values...