The Character Of Protease Produced By Bacillus Licheniformis And Its Application In The Steeping Technology Of Corn Wet Milling

Bacillus licheniformis is one of the most important bacterial strains with the ability to produce alkaline protease. The strain in our lab has low yield of alkaline protease, but it is sensitive to the factors of mutagenesis. Therefore, it has a lot of the potential to be improved.The protein surrounds the cornstarch granules, and it is an essential process to degrade the protein during starch produce industry. The presence of peripheral endosperm that acts as a barrier against the penetration of the sulfur dioxide solution, a harder protein matrix and cross-linking, which enrobes starch granules thus decreasing starch yields and quality. During the process of machining cornstarch, in addition to digest the protein which wrapping the cornstarch, the addition of sulfur dioxide is required in considerable amounts, and this chemical is an environmental concern. The overseas scientists had attempted to add enzyme to the steeping water when produced starch with the improved process in lab, and had received a perfect effect. But there was also a problem that it cannot be practiced in the industrial production because of the high cost. In this paper, on the basis of conventional processing technology of corn wet milling, we used the improved process in lab and used the character of protease which can digest the protein to study a new approach that is economical and environmental protection.Thus, the aim of this paper was to screening a strain of microorganism which has the ability of high yield protease and high enzyme active; then adding the fermentation liquor which has high yield protease to the steeping water to achieve the motive for shortening the steeping time, depressing the concentration of sulfur dioxide while maintaining high product yields and quality.In this paper, taking the Bacillus licheniformis as the initial strain, its enzyme activity was 1103 U/mL. The mutagenesis treatments on protoplasts were used. All factors effecting on formation and regeneration of protoplasts were studied. These factors include penicillin, lysozyme, glycin and stabilizers. Under the optimum conditions of protoplasts formation and regeneration, the protoplasts from the initial strain were prepared. The optimum conditions were confirmed: 1mg/mL glycin, 0.4u/mL penicillin; 2mg/mL lysozyme, temperature 37℃for 6 hours. The rates of formation and regeneration of protoplasts of the strain had reached to 93.6% and 36.5% respectively. Then, the ultraviolet radiation mutagenesis treatments on protoplasts were studied. The optimum conditions were as follows: The dosage of ultraviolet radiation was 3 minutes(simple treatments).After the second mutagenesis treatments on protoplasts of the strain and a large amount of the regenerative mutants, a stable mutant number 16 producing high yield alkaline protease was obtained. The enzyme activity was 3073U/mL.The fermentation conditions of number 16 strain which produced high alkaline protease in flask were studied. The optimum fermentation conditions were confirmed: seed age 12h, inoculum volume 2%, a 250mL flask containing 25mL medium,210r/min,30℃for cultivating 44 hours.Enzyme is a kind of protein which can lose its activity easily. Hereby, it is very important to maintain its stability during separation and purification. The alkaline protease was purified from the fermentation liquid of Bacillus licheniformis by means of ammonium sulfate salt out and dialysis followed by DEAE-Sephadex-A-50 anion exchange column chromatography,CM-Sephadex-C-50 cation exchange column chromatography. The purified protease was demonstrated to be electrophoretic homogeneity by SDS-PAGE, with a molecular weight of 28.5kDa. The properties of the alkaline protease were studied. The optimum pH and temperature toward the hydrolysis of casein were pH10.0 and 55℃.The alkaline protease was stable in the range of 40-60℃, and in the pH range from 5-11. Also, it can maintain its enzyme activity on SDS. Ag2+ and Cu2+ restrained the activity of protease, but Ca2+ and Mg2+ had a little activation on the protease. All the character were mastered had the effects for the future experiments.It is very firm about the combining of the starch and the protein in the endosperm of maize, and the protein surrounding the cornstarch granules. For the purpose to release the starch, breaking the connection between them is necessary. It needs a large amount of sulfur dioxide solution in the conventional steeping to degrade the protein and it can assist in the separation of starch and protein while milling the kernel. Although there are many advantages in using sulfur dioxide, but the most serious problems are the cauterizations of the equipments, the pollution of water under the ground, the rudimental sulfurous acid in the products, etc.The studies of scholars had confirmed that the application of enzymes to the normal steeping step of wet milling was not an effective means of decreasing the steeping time or sulfur dioxide usage. It was likely that the enzyme did not adequately penetrate the intact kernel and therefore cannot degrade the starch-associated protein. The conclusion of the optimum conditions about the protease fermention liquid from the results of Analysis of orthogonal experiment and SSR test of each treatment were: Maize were steeped for 18 hours with the solution of 0.1% sulfur dioxide and 0.5% lactic acid; then separated the germ and steeped for another 6 hours with 20% protease fermention liquid. The overall steeping time with protease fermention liquid and the improved method of two-stage procedure was 24 hours while the conventional process was 36 hours. And the yield of cornstarch was 67.79%,higher than the yield of convention method simulated in lab about 9.5 percents. The modified process and method greatly decreased and possibly eliminated the need for sulfur dioxide addition, while producing starch yielded and quality equivalent to that from the conventional process for the future.