Isolation Of Phytase-producing Microbes In The Soils Of Tibetan Plateau And The Phytase Proferties

Phytase is a kind of enzymes that stepwise hydrolyse phytic acid to release phosphate radicals and inositol derivatives. As a new type of feed additives, it has great application potential in the areas of animal nutrition and beneficial to environmental protection. Monogastric animal feed supplemented with microbial phytases effectively improves phytate phosphorus utilization, reduces the excretion of phosphorus in animal manure and improves the nutrient value by removing the anti-nutrient factor of phytate in intensive livestock production. This field has been the research focuses domestically and internationally.Many previous studies indicated that there was a diverse and rich microbe in the soils of the Qinghai-Tibet Plateau. The investigation aims of this study to screen the phytase-producing microbial strains from extreme environment soil samples and then further to select for high yield strains by mutagenesis, elucidate the enzymatic property of phytase produce by the isolates. Further, the potential application of the phytase in the feed additives was exploited. This study will provide prospects in the feed additives of animal husbandry. The main results are as below.?1? From the soil samples that were collected at 12 sites along with Qinghai-Tibet highway, we isolated 18 phytate degrading microbes. Among all isolates, 55.5% were screened from the rhizosphere soils of shrubs in Qaidam Basin, 27.8% from the sites along Tibetan Plateau railway, 16.7% from the desertification grassland of Tibetan Plateau. By sequencing the 16s/18 s rRNA gene, 18 different species were identified and named, among them, 4 isolates, Erwinia persicina AT1-1, Fusarium tricinctum BLH-2, Talaromyces pinophilus NMH 2-1-1, Cladosporium cladosporioides NMH 3-3-2, are the first report as phytate degrading microbies. Through the assay of phytase activity of the 18 strains, Penicillium glabrum NMH 2-3-1 displayed the highest capability of phytase production?2695.3 U/m L?.?2? The phytase properts produced by Penicillium glabrum NMH 2-3-1 were measured under defined conditions. This phytase has stability at pH 4.0-8.0 and temperature 20-60?. The optimum temperature is 55?, and under 60? for 1h incubation, the enzyme activity remained by 75%, suggesting the good thermal stability. Mn²⁺, Cu²⁺, and Al³⁺ strongly inhibited the phytase activity, while Ca²⁺ and Mg²⁺ enhanced the phytase activity. The enzyme also showed good resistance to hydrolysis of pepsin and trypsin.?3? The high phytase-producing P. glabrum NMH 2-3-1 was induced mutation with UV radiation and 4 higher phytase-producing strains were selected, that is, P. glabrum U1, P. glabrum U2, P. glabrum U3, and P. glabrum U4. Among them, P. glabrum U3 can produce the highest phytase activity of 3611.3 U/m L. The enzyme activity was increased by 34% in compared with the original strain. The strain P. glabrum U3 with the highest phytase activity was further induced mutagenesis using EMS. After the screening procedures, we finally selected a strain P. glabrum E1 which can increase the phytase activity by 8.9% and 49% than that of P. glabrum U3 and the original strain, respectively. The abilities of producing phytase were stable after 6 generations.?4? Plackett-Burman and Box-Behnken design were used to optimize the technological conditions of phytase production of P. glabrum E1. The results showed that the concentrations of NH₄NO₃, MnSO₄, and FeSO₄ in the medium were the three key factors that influence the phytase activity. Finally, the optimal fermentation medium composition was: 1.5% starch, 0.375% NH₄NO₃, 0.05% MgSO₄·7H₂O, 0.05% KCl, 0.008% MnSO₄, and 0.0078% FeSO₄. The optimal enzyme-producing conditions were: pH 6.0, 2% inoculum volume, 30? of fermentation temperature, and shaking speed of 160 r/min. Under this optimized technological conditions, Phytase activity produced by P. glabrum E1 was up to 4197.15 U/mL.?5? The activity of the rude enzyme liquid in degrading phytate phosphorus in the feedstuff was measured. The results showed that the contents of phytate phosphorus in cotton pulp, wheat bran, soybean meal, and corn flour were 69.64%, 62.82%, 61.24%, and 62.82%, respectively. After 3 h hydrolysis of the phytase at 37?, the phytate degradation rate in above 4 feedstuffs were 47.08%, 59.95%, 62.48%, and 65.74%, respectively, showing the best effects of the phytase on phytate degradation in cotton pulp, followed by the order of soybean meal > bran > corn flour.