Studies On The Genetic Diversity And Thermophilic Keratinase And Lipase Of Thermophiles From Hot Springs In Tengchong Yunnan

Enzymes produced by thermophiles and hypermophiles are typically thermostable and thermophilic.Basing on these important advantages, thermoenzymes are attracting much more industrial interest.The composition of the microbial communities in theromphilic environment has provided the base of isolation and purification of thermophilic strains and enzymes.The investigation of the structure of stability will illuminate the structural basis of the stability and catalytic mechanism at high temperature.This knowledge can lead to the development of new and more efficient protein engineering strategies and a wide range of biotechnological application.This thesis includes several parts as follows:1.Genetic diversity of thermophilies of hot springs in Tengchong,YunnanThe cultured thermophilic strains may be represented a small part of community in environment using traditional isolation methods.The advent to cultivation independent methods has provided researchers with the ability to determine the composition of microbial communities and identify numerically important,but not yet cultured organisms.We used molecular ecological methods to study the phylogenetic relationship and ecological diversity of thermophilies in sediments in hot springs in Tengchong,Yunnan.We selected 7 samples of soil sediments from hots springs in Tengchong,Yunnan,and used cultivation independent methods to study the genetic relationship.Phylogenetic analysis of the 16S rDNA sequences with the template of total soil DNA suggested that thermophiles in nature are much more diverse than were ever known,including thermophilic bacteria and archaea.The thermophilic members were distinct in each hot water environment and the compositions might be strongly influenced by the environmental conditions.Comprehensive analysis of 16S rDNA sequences were a lot of numbers of thermophiles in nature,while we can only isolated a small number of strains using traditional methods.It is suggested it was more different in diversity between composition communities of thermophiles and metabolically active communities of thermophiles in soil environment.These results should also be helpful of giving insight into the nature of unidentified bacterial and archaeal lineages of theromphilic organisms and offer a promising strategy to applications in industrial processing of thermophilic strains and enzymes. 2.Isolation of a new thermophilic,native-feather-degrading bacterium and characterization of the thermophilic keratinaseKeratin is a highly stable and insoluble structural protein found in feathers and wool.Feathers are mainly composed of pure keratins,which are not easily degraded by common proteases.There are plenty of resources of feather keratin in the world. There is an abundance of potential proteins and amino acids accumulate in livestock and poultry feathers,which could be the source of excellent feedstuff proteins.It is the key to find an appropriate way to degrade keratin in this study,as the keratin is very difficult to be decomposed due to the steady configuration.Traditional treatments also result in environmental pollution,and therefore,the biodegradation of feathers has attracted much interest.Different from conventional methods such as physical chemical degradation,biodegradation holds outstanding effect and no defects like low efficiency,high expend and excessive contamination,which not only make use of the protein source but also help to protect environment.As the applications of keratin and keratinase to all kinds of industry emerge more,those are considered to be provided with more values of economic and social,and with brilliant prospet.A thermophilic,native-feather-degrading aerobic bacterium was isolated from a geothermal hot spring in China.The bacterium(strain RD-2) was identified as a member of the genus Geobacillus based on its morphological and biochemical characteristics and 16S rDNA sequence.The strain was able to degrade native feathers,with optimum growth at 60℃and pH 7.5.The culture supernatant of strain RD-2 was active on keratin azure,a keratin substrate,and exhibited optimum activity at pH 8.0 and 75℃.The purified keratinase is a monomeric enzyme with a molecular mass of 65 kDa.Additionally,we found that in the presence of the reducing agent DTT,the activity of the keratinase from strain RD-2 increased two-fold.Therefore,the keratinase of Geobacillus sp.RD-2 is likely a promising enzyme for application in industrial processing.3.Characterization of thermophilic lipase lipGRD and identification of residue Tyr224 critical for the temperature dependencyIn recent years,thermophilic lipases from thermophiles have been specially focused due to their inherent thermoactivity and thermostability.These characters confer high thermal stability as biocatalysts derived from thermophiles prevailing in various industrial processes.We cloned a lipase(lipGRD) gene of the Geobacillus sp. RD-2 and expressed the protein(lipGRD) in Escherichia coli.His-tagged lipGRD was purified by heat treatment and Ni-NTA affinity.Molecular size of purified lipGRD by SDS-PAGE analysis was 43 KDa,which was in agreement with predicted protein size,and the enzyme had an optimum temperature of 55℃and and an optimum pH of 7.5,respectively.The enzyme was highly thermostable.Initially,we obtained a PCR product with mutated residues because we used a Taq DNA polymerase in PCR reaction.Surprisingly,we found that the mutant protein lost the thermostability of the wild type.Mutants Y224C,and Y224P showed similar activity as that of the wild-type lipGRD at optimum pH of 7.5,but displayed distinct temperature profiles.Mutant Y224C was optimally active at 35℃,whereas Y224P was optimal at 65℃and its thermostability was increased significantly.Based on the structure of lipase from Geobacillus stearothermophilus L1,we got the the structure model of lipGRD.It is proposed that high temperatures might have induced a temperature switch of the lid helix(α6) and exposed the active site for catalysis and the extra helices(α7-α9) near the lid helix(α6) of the L1 lipase,seems to contribute to the stability of the enzyme,which may lead to decreased thermoactivity and thermostability.The extra helices(α7-α9) are not present in thermal unstable lipase.Notably,the residue Y224 is located at the region of helixα7, which is just downstream of the lid helix.For the lipGRD/Y224C mutant,the form of disulfram with Cys and a Cys at site 64,might changed the conformation of the lid helix,and made the lid open at low temperatures in the presence of lipid substrates, which may lead to decreased thermostability.However,the interaction of lipY224P with several adjacent Pro may improve the hydrophobic interaction between the helixα7 and the lid helixα6 and strengthen the tight packing of the active residues,which increased thermostability.In summary,our results highlight the significance of the diversification of the lipase structure basing on the point mutation,and offer a promising strategy to regulate the thermostability of lipase for industrial uses.