Isolation Of Thermophilic Prokaryotes And Screening Of Thermostable Xylanases From Hot Springs In Tengchong Of Yunnan Province,China

Hot spring is a stable and close microbial ecosystem. The high temperature of hot spring leads the microbes to adapt a unique physiological mechanism and evolve the specific genes, which not only help them to adopt to high temperature but also mark them as a potential source of several enzymes with unique features that have industrial significance. Among various industrial significant enzymes, xylanases is of great importance due to its widespread applications.Endo-β-1,4 xylanases (EC 3.2.1.8,1,4-β-D-xylan sylanohydrolase) has engrossed much attention and has been extensively employed in food, animal feed, textile, waste treatment, and bio-fuel industries. Many commercially important xylanases are available in the market, however, due to lacking thermo-stability they can’t meet up to the industrial demands. Therefore, there is demand for xylanases with thermo-stability. Understanding the current situation, the present study is an attempt to understand the microbial diversity of hot springs located in Tengchong county of Yunnan Province in west-south China by using culture-dependent technology and metagenomic sequencing along with the screening for novel isolates with thermo-stable active xylanases and its genome analysis.Eight hot springs from Tengchong country and thirteen isolating media were designed to isolate the prokaryotic thermophiles. The functional diversity of thermophilic microbial community was studied using Biology-ECO microplates. Further, the metabolic difference were investigated by using different carbohydrate substrate, Shanno and McIntosh index, PCA and cluster analysis to better understand the microbial community’diversity and the metabolic characteristics.A total 634 isolates were obtained from eight hot springs, belong to five phylums, seven classes, eleven orders, nineteen families, thirty two genera and ninteen potential belongs to novel taxa. The isolates were classified into three metabolic types which are affiliated to Firmicutes, Proteobacteria, Deinococcus-Thermus, Actinobacteria and Chloroflex.The 634 isolates were screened for thermostable xylanase activity. The ratio of isolates with xylanase activity was 9.1% which was very low and hence we developed various enrichment strategies. In first enrichment strategy,15ml sample of hot spring was injected into the concentrated media, which were taking the xylan or native substance such as bran, corncob and sawdust from beech-wood as the sole carbon source whereas in second enrichment strategy, bran, corncob and sawdust from beech-wood was added into hot springs for 3 months and then incubate these substances with cultured media for 1 month in lab. The results showed that the ratio of xylanase activity of thermophilic prokaryotic microbes from enrichment treatment 1 and 2 was 16.3% and 45.7% respectively which was much higher than earlier method.Among 634 isolates, the isolate Geobacillus sp. YIM 71344 was potential xylanase producer which was selected for further studies. The results showed that the isolate Geobacillus sp. YIM 71344 produced xylanase activity up to temperature 70 ℃ being optimum at 60 ℃. The pH range for xylanase production was 5.0-12.0, with optimum at 6.0 and pH 9.0. These results indicate that strain Geobacillus sp. YIM 71344 could produce thermostable and high pH tolerant xylanase.Genome sequencing of strains Meiothermus sp. YIM 71147 and Geobacillus sp. YIM 71344 was carried out. The data of genome was assembled and the genes were predicted, classified and the function of genes was annotated. The results showed that there are many genes associated with the degradation of xylan. The xylanase gene named xynGL001395 was detected in strain Meiothermus sp. YIM 71147 while xylanase genes named xynGL002415 and xynGL002429 were detected in strain Geobacillus sp. YIM 71344. Analysis information about these three genes of xylanse indicated that they all belong to GH10 family with the typical (α/β) 8 structure.Metagenomic analysis of Jinze (JZ) and Gongxiaoshe (GXS) hot springs from Tengchong country was performed to better understand the diversity of thermophilic community and to screen the xylanse genes. The results showed that the domain thermophiles in JZ hot spring included bacteria and archaea, and the domain thermophiles in GXS was bacteria. Firmicute and Proteobacteria were prevalent in two hot springs. Forty-four xylanase genes were screened from JZ hot spring, and 85% percent of them possessed novelty. Eighteen xylanase genes were screened from GXS hot spring and all of them possessed novelty. Majority of the xylanase genes from two hot springs belong to GH10 family.This work will serve as groundwork to exploit the novel products including thermo-stable enzymes and other bioactive compounds with special characteristics produced by thermophiles, as well as to develop the potential biotechnological applications in various industrial fields.