| The majority proteins used in food industry was vegetable protein, but, due to their poor solubility, some of them was limited in application, such as soybean protein, oryzenin and so on. Therefore, we could carry out different methods to modify proteins to improve their performance and functional characteristics.Protein deamidation was one of the most useful method to modify proteins. And we could use different methods to finish deamidation, including physical method, chemical method and enzyme method. Due to their own limitations, using the former two methods, the process of deamidation always accompanied by a variety of negative side effects. To sum up, enzyme method was still the most attractive one.Protein glutaminase (PG, EC 3.5.1) was a noval deamidation enzyme found in recently years. Compared with other deamidation enzyme, it had unique advantages, such as, its substract was protein but little peptide.Therefore, PG could catalysis protein to deamide without protein hydrolysis pretreatment. At the same time, its specificity was very high, only catalysised glutamine amide group, instead of asparagine. And it couldn’t bring bad flavor.Therefore, PG could stand out on application in the food industry.But due to the low enzyme production ability of wild strains, the progress of industrial development is limited greatly.This research was amid at obtaining more strains producing PG by screening from soil. And we analysis the taxonomic status of isolates and identified PG producing by these isolates. Finally, we optimized the fermentation medium of isolates to improve the PG production.The results of this research were as follows:First, we screened PG producing strains from 799 soil sample collecting from different regions of China. The relationship between soil characteristics, such as seosons, environment of soil and pH of soil, and positive rate of soil samples was established. Totally, we achieved 206 isolates producing PG through screening from soil, so the efficiency of screening was quite high。The result demonstrated that the soil sample collecting in spring was more easily than other sonsons to isolate strains producing PG, and the rate of obtaining isolations from farmland, forest, farm, and the water was higher than others. It seemed that we scucceeded more easily when screened from the soil that pH was ranging from 8.0-9.0.Second, we selected 36 isolates to identify in morphological, physiological, biochemical test and molecular biology (16S rDNA).The results have shown that 32 of them was identified to Chryseobacterium proteolyticum; strains QRD04862、 QRD12462、QRD121161 was belong to Chryseobacterium indologenes; And strain CMJ01105 was Chryseobacterium culicis. And nine in ten of the isolates was Chryseobacterium proteolyticum, which was not forbidden in food industry. Then, PG produced by different isolates were identified. And the DNA sequence homology of PG produced by isolates and strain 9670 was as high as 98%. And the deduced amino acid sequence of PG from isolates was same to strain 9670 completely.Finally, we preliminarily optimized the fermentation medium of isolates. Through one factor experiments, we selected the best carbon source and nigton. Exploring the optimical concentrate of CaCO3 to stabilize the pH of fermentation system. Following, we combinated Plachett-Burman design and Box-Behnken Design to obtain the optimal fermentation medium system. We tested the optimal fermentation medium and the results proved that producing enzyme ability of isolates was increased 247%. |
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