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Extremely thermostable and acid-stable a-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE. The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L. The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95 degrees C and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0 - 7.0 for 48 hours. About 60% of the initial enzyme activity was measured after 1h of incubation at 110 degrees C. The activity was strongly inhibited by Fe2+, Cr2+ and Cu2+, While Ca2+ had no effect on it. DTT and EDTA had no effect on the activity.

The study used the functional Lactobacillus acidophilus producing α-amylase, protease, phytases and β-mannanase to carry out cooperative fermentation of soybean meal. The effects of the inoculating amount, fermentation temperature, fermentation time and the ratio of water to material on the content of acid-soluble protein were studied by the single factor experiment. On this basis, through Box-Behnken design experiment, the quadratic regression model was established, and the optimal fermentation conditions were the inoculated amount of bacterial liquid of 12.3%, the fermentation time of 78.9 h, the fermentation temperature of 36.8 °C, and the ratio of water to material of 0.9 L/kg. Under these conditions, the crude protein content increased by 15.13%, the acid-soluble protein content increased by 66.91%, hemicellulose content decreased by 24.93%, the free amino acid content was 11.62 times higher before fermentation, the trypsin inhibitor content decreased by 91.68%, the urease activity decreased by 95.67%, the phytic acid content decreased by 83.87%, and the viable bacteria count in fermented soybean meal was 1.37x1010 CFU/g. The study indicates that the composite strain fermentation mode can effectively improve the nutrient composition of soybean meal and remove antinutrient factors, so as to improve the utilization rate of soybean meal and provide a reference for the industrial production of fermented soybean meal. [ABSTRACT FROM AUTHOR]