Isolation And Identification Of Thermophiles From Changbai Mountains Hot Springs And Characterization Of A Novel Thermophilic Lipase

The researches on thermophilic microorganisms and enzymes which have great impact on theoretical study and industrial applications have always been hot objects since 1990s'. Changbai Mountains exhibit an aechaeo-ecological system because of its vulcanian geology and special geography environment, with many hot springs possessing redundant thermophilic microorganisms and gene resources. This paper investigated the diversity and evolution relationship of culturable thermophilic microorganisms from Changbai Mountains'hot springs, and several novel thermophilic species were isolated and identified. Then, the lipase prospecting primers were designed, and a fragment of lipase gene was obtained from hyperthermophilic F. changbaicum CBS-1 chromosomal DNA by PCR amplification with the lipase prospecting primers. The full-length gene of lipase was obtained by using genome-walking method, and then the lipase gene were cloned and successfully overexpressed in E.coli. Finally, we characterized the recombinant lipase and probed into the stability mechanism of the novel thermophilic lipase.Changbai Mountains'hot springs are neutral (pH of water, ~7.2) and rich of metal ions and acid radicals. Microorganisms from hot springs samples were cultured by plating on four kinds of enrichment mediums. Through classic culture and gradient dilution culture, nine thermophilic strains were obtained, named as CBS-1 to CBS-9. To determine the taxonomy position of the newly isolated thermophilic bacterium in evolution history, 16S rRNA sequence homologous alignment and phylogeny analysis were performed between them and related species. The results showed that they belonged to three genera from two different eubacterial families, indicating the extensive microorganism diversity of Changbai Mountains'hot springs.According to the 16S rRNA sequence homology analysis, at least three strains (CBS-1,CBS-4 and CBS-5 ) among the nine newly isolated ones were potentially novel species and the strains CBS-1 and CBS-4 were studied by using polyphasic taxonomy methods. The strain CBS-1 could grow at 90°C and the optimum growth temperature was at 70-80°C which was the highest compared to the other Fervidobactium species that can barely grow at 80°C. The optimum growth temperature of the strain CBS-4 was at 70°C which was similar to other Fervidobactium species. The G + C content of strain CBS-4 was the lowest compared to the other species within the genus Fervidobacterium (32-40 mol %). The low (G+C) content of the two strains probablely implied the unique ecological system of Changbai Mountains'hot springs. On the fatty acids component aspect, the high concentration of C16:0 in the fatty acids profile of the two strains'cell envelope supported their inclusions as two members of the genus. However, the proportions of the C16:0 in the strains CBS-1 and CBS-4 were both lower than the other species. Furthermore, the DNA-DNA hybridization result exhibited the less 70% hybridization values between either CBS-1 or CBS-4 and the other Fervidobacterium species. The results of genotype, phenotype and chemical taxonomy indicated that the two strains were not related at species level to the other Fervidobacterium species. Based on the data of polyphasic taxonomy, we proposed to assign strain CBS-1 T and CBS-4T to two novel species of the genus Fervidobacterium, for which we proposed the names Fervidobacterium changbaicum sp. nov. and Fervidobacterium tianchisum sp. nov.. The type strains were strain CBS-1 T (=DSM17883T=JCM13353T) and CBS-4T (=JCM14752T), respectively.For prospecting novel lipase genes from the novel isolated hyperthermophilic Fervidobacterium changbaicun, we aligned the all sequences of the known lipases and classified the two conserved regions of lipases according to the sequence similarity. The lipase prospecting primers were designed by using CODEHOP (Consensus degenerate hybrid oligonucleotide primers) primer design principles, then a fragment of lipase gene was obtained from F. changbaicum chromosomal DNA by radom combination PCR with the lipase prospecting primers. The full-length gene of lipase was obtained by using Genome walking and nested PCR methods. The sequence alignment on NCBI research showed that the novel lipase possessed very low homology with known lipase sequences, and the sequence similarity was all less than 30%. The lipase gene was cloned and successfully expressed in E.coli. The novel lipase (FCLip1) was the first lipase from hyperthermophiles and also the first characterized lipase from the family Thermotogaceae, respresenting a slowly evolving lineage within the bacterial domain. Subsequently, we characterized the novel lipase on enzymology and probed into the stability mechanism. The results exhibited that the optimal temperature and pH for FCLip1 was 78 oC and 7.8 respectively. FCLip1 exhibited high activity and wide substrate chart towards p-nitrophenol esters and triglycerides. Among tested p-NP esters and triglycerides, p-nitrophenyl caprate and trilaurin was the most preferential substrate of the FCLip1 respectively. The specificity constant, kcat/Km value increased with increasing chain length, reaching a maximum with trilaurin and p-nitrophenyl caprate, then the kcat/Km value decreased with increasing chain length. For the p-nitrophenol esters substrates, Km value decreased with increasing substrate acyl chain length which indicating that FCLip1 showed high affinity towards long chain p-nitrophenol esters. On the contrary, Km value decreased with increasing substrate acyl chain length which indicating that FCLip1 showed high hydrolysis rate towards short chain p-nitrophenol esters from the catalytic reaction point. For the triglyceride substrates, though the kcat and Km values fluctuated irregularly with increasing acyl chain length, the Km for trilaurin was a minimum with 12.11μM which indicating that FCLip1 showed highest affinity towards trilaurin.We studied on the stability mechanism proceeding with thermostability, pH stability, Guanidine hydrochloride (Gdn-HCl) unfolding and theoretic analysis on primary structure. FCLip1 possessed very well thermostability at less than 70 oC. The half life of 70 oC was about 8.5 hours, which was higher than thermophilic lipases from Bacillus (Geobacillus). The results from thermal denaturation and pH stability studies showed that the tertiary structure and second structure did not change coorperatively. The tertiary structure of enzyme was destroyed much faster and more serious than its second structure. FCLip1 exhibited greatly well pH stability ranging from pH6.0 to pH10.0. The exposure of hydrophobic residues of the enzyme molecule was the minimum at pH7.0 by ANSA fluorescence detection. However, the optimal pH for catalytic reaction and conformation stabilization was pH8.0. These results indicated that appropriate exposure of hydrophobic residues was propitious to catalytic reaction and conformation stabilization. The concentration of Gdn-HCl at the transition midpoint (Cm) was 3.65M and the free energy (△G) was 33.57KJ/mol during Gdn-HCl unfolding. The activity conformation of enzyme was destroyed much easier than its tertiary structure by determining the effect of Gdn-HCl on catalytic activity and intrinsic fluorescence of FCLip1. The partial unfolding was preexistent during the unfolding course. In addition, the unfolding was reversible at less than 4.0M Gdn-HCl concentration, and the recovering rate was inversed to Gdn-HCl concentration, indicating that the recovering of enzyme's conformation was nearly relative to the denaturant concentration.In this paper, for the first time, we isolated and identified two new hyperthermophiles from Changbai Mountains hot springs and characterized a novel thermophilic lipase from hyperthermophiles. This work will contribute to the development of extremophile molecular enzymology and engineering.