Studies On Species Diversity And Thermophilic Enzyme From Hot Spring In Part Regions Of Yunnan

Thermophilie microbe and other microbe living in extreme environments increase the diversity of life. These extreme microorganisms are important in the study of scientific theory and application. Thermophilic enzymes are superior to mesophilic enzymes in many aspects. There are many hot springs in Yunnan Province, in which thermophilie microorganisms are abundant. In this study, soil and water samples were collected from25hot springs and neighboring soil distributed in Dali, Lincang, Dehong, Baoshan and Honghe region of Yunnan Province. Bacteria and fungi were isolated from these samples, and then were identified. In order to exploit thermophilic enzyme and further study their mechanism of stability, a thermophilic strain and two mesophilic strains of Trichoderma harzianum were selected. The characters of thermophilic and mesophilic proteae, cellulase, xylanase, chitinase were compared respectively. Results as following:1. Isolation and identification of thermophilic bacteria.106strains were acquired by the pure culture, including8species:Bacillus licheniformis, Bacillus teqilensis, Bacillus subtilis, Bacillus altitudinis, Bacillus axarquiensis, Bacillus flexus, Brevibacillus agri, Brevibacillus brevis.2. Isolation and identification of thermophilic fungi.79strains fungi were acquired by the pure culture, which were identified by combing morphological characters and ITS sequence, including8species:Myceliophthora thermomyce, Rhizopus microosporus, Rhizopus pusillus, Rhizomucor miehei, Rhizomucor pusillus, Scytalidium thermophilum, Trichoderma harzianum, Thermomyces lanuginosus. Among them, S. thermophilum is an dominant species, representing57%of the sample. Rhizopus microosporus, Rhizopus pusillus were first recorded as thermophilic fungi after these strains were tested their growth temperater.3. Part characters of Thermophilic and mesophilic enzymes. Thermophilic and mesophilic proteae, cellulase, xylanase, chitinase produced by T. harzianum thermophilic strain T1, mesophilic strains YMF1.403, YMF1.408were compared from including thermostability et al. aspect(1) The optimal fermentation parameter of thermophilic cellulase are50℃,180r/min,9days, while those of normal temperature cellulase are28℃,160r/min,7days. The optimal temperature and pH for the thermophilic cellulase activity was50℃and4.8, and activity began decline when pH was more than5.5. The residual activity was up to60%after the enzyme was incubated at70℃for30min. The optimal temperature and pH for the mesophilic cellulase activity was5.5and50℃, and activity began decline when pH was more than6.0. The residual activity was less than50%after the enzyme was incubated at60℃for30min.(2) The optimal fermentation parameter of thermophilic xylanase are45℃,180r/min,7days, while those of normal temperature xylanase are28℃,160r/min,7days. The optimal temperature for activities of the xylanase from all three strains was50℃. The activity of thermophilic xylanase was stable when temperature was between45℃and75℃, and the residual activity was40%when the temperature is up to80℃. The activity of mesophilic xylanase was stable when temperature was between45℃and55℃, and the residual activity was20%when the temperature is up to70℃.(3) The optimal fermentation parameter of thermophilic protease are50℃,180r/min,6days, while those of normal temperature protease are28℃,160r/min,7days. PMSF shows a strong inhibition to all three kinds of proteases. The activity of thermophilic protease remained above80%when temperature was between4℃and60℃. The activity of normal temperature protease was stable when temperature was between4℃and50℃, but the residual activity was less than40%when the temperature was up to50℃. The optimal pH for thermophilic protease and mesophilic protease was7.0and8.0respectively.(4)The optimal fermentation parameter of thermophilic chitinase are45℃,180r/min,7days, while those of normal temperature chitinase are28℃,160r/min,7days. The optimal temperature for activities of the chitinase from all three strains was 50℃. The activity of thermophilic chitinase remained above80%when temperature was between4℃and60℃. And the residual activity of mesophilic chitinase remained only20%when the temperature was up to50℃.