Thermophilic Diversity From Hot Springs In Houqiao Of Yunnan Province And Screening Of Cellulose-degrading Bacteria

BackgroundAn abundant and unique group of thermophilic microbes were found to exist in hot spring environment.Major of them were belong to prokaryotes.These thermophilic microbes evolved to adapt to their extreme environment due to the prolonged exposure to high temperatures.The bio-structure,physiology,genetics and adaptation mechanisms of thermophiles from hot spring evolved and could produce a variety of thermophilic enzymes and other special biologically active substance.Cellulose degrading enzyme system mainly consists of cellulase and xylanase.They have a wide range of applications possibilities,but their low-thermal stability limits their application.One of effective way is to obtain abundant thermophilic bacteria from hot springs with extreme high temperatures,and then to screen thermostable enzyme of degradating the cellulose which was produced from these thermoplic bacteria.ObjectiveIn order to explain the thermophilic microbes group composition and species diversity of Houqiao hot springs in Yunnan Province,a suitable depend-culture method including temperature range,isolation media was established to obtain abundant thermophilic bateria.Isolates with xylanase and cellulase activity were screened with Congo red staining.The xylanase gene from thermophiles was cloned and expressed,the thermostable xylanase protein was purified,and the characteristics of thermostable xylanase protein was studied.This research is aim to established a suitable isolation system for Houqiao hot springs environment,obatain numerous thermiphilic bacteria and screen thermophilic isolates with xylnanse-or cellulose-activity,as well as studied the thermostable xylanase produced from thermophile.As a result,this research provides a large number of thermophilic bacteria for function-screening such as xylanase,cellulose and other function traits,studying on thermostable xylanase will futher expands the application of xylanase in industrial aera.MethodsIndepend-culture study was carried out for community composition and species diversity of thermophilic microbes from Houqiao hot springs by using High-throughput sequencing technique.Depend-culture study was preformed by establishing a suitable isolation system,including 7 isolation media and 3 isolation temperatures.Thermostable ylanase gene was cloned and heterologously expressed by molecular biology technology.Thermostable xylanase protein was purified and the enzyme properties was studed for further application.Results1.The community composition and species diversity of thermophilic microbes from Houqiao hot springs environment was analyzed by high-throughput sequencing technique.Of the total 18 samples,830,022 original sequences were obtained,belong to 65 phyla,139 classes,236 orders,376 families,603 genera.The dominant groups at phylum level were Chloroflexi.2.A total of 1,342 isolates were obtained from eleven hot springs,belong to six phyla,twelve classes,twenty-four orders,thirty-seven families,sixty genera and eighty-nine potential belongs to novel taxa.The dominant groups at phylum level were Firmicutes and Deinococcus-Thermus.Through screening,71 strains of thermophilic prokaryotic microbes with xylanase activity and 30 strains with cellulase activity were found.3.Genome sequencing of Thermus sp.CFH 73267 was preformed and analyzed a xylanase gene sequence of the tenth family(GH10)was obtained,which was cloned and expressed in E.coli and thermostable enzyme protein was puried and its enzymatic properties was studied.The results showed that the protein of xylanase was super-thermostable,the optimum catalytic temperature and pH of this enzyme were 95? and pH6.0,respectively.After incubation at 80? for 200 min,the enzyme activity could still be maintained at more than 80%.The optimum reaction substrate was oat xylan.ConclusionsThere are abundant thermophilic prokaryotic microbes in Houqiao,Yunnan Province.This work will serve as groundwork to exploit the novel products including thermostable enzymes and other bioactive compounds with special characteristics produced by thermophiles,as well as to develop the potential biotechnological applications in various industrial fields.