Screening, Thermal Adaptation Of Marine Thermophiles And Characterization Of A Thermal-stable Lipase

High temperature is a characteristic of prehistoric earth surface. A biological essential feature of primitive life is the adaptability to hot environment. The microorganism, as the oldest life that formed on the earth, has a particularly surprising high-temperature adaptive capacity. When the surface temperature of the earth is reduced gradually, various mesophiles appeared, and thermophiles may be the products of mesophiles adapted to the high-temperature environment.Because themophiles have great application values and developmental prospects on genetic engineering, protein project, fermented project, and development and utilization of mineral resources, thermophiles have attracted extensive studies in the last few years.In this study, 50 strains of thermophilic bacteria were isolated from deep-sea hydrothermal vents in Pacific and a hot spring in Xiamen of China. Moreover, lipase-producing thermophiles were obtained, in which the strain TW-1 presented the highest lipase activity. Based on 16S rRNA sequence, TW-1 was assigned to Gebacillus sp. Subsequently, cloning and expression of a 1254-base lipase gene were conducted using the strain TW-1 in Escherichia coli. The physical and chemical properties of recombinant lipase were analyzed. The results showed that the optimum temperature for recombinant lipase was 40℃. The recombinant lipase has an optimum pH at 7-8, keeping 80-100% of the activity within the range of pH 6.0-9.0. Under the conditions of 1mM inhibitors (EDTA, 2-ME, SDS, PMSF or DTT) and 0.1% detergents (Tween 20, under Chaps or Triton X-100), EDTA, 2-ME, SDS could partially inhibit 18-30 % of the recombinant lipase and crude enzyme activities. It had not been influenced by DTT and PMSF. Tween 20, Chaps and Triton X-100 had almost no influence on the crude enzyme, however, they had about 5-10 % inhibition on the recombinant lipase. At the presence of 1mM or 5mM of Ca²⁺, Mg²⁺ or Zn²⁺, or 1 mM of Fe²⁺ or Fe³⁺, the activities of both enzymes increased to 103-283 %. 1 mM of LiCl, CuCl₂ or MnCl₂ had a slight inhibition on the enzyme activity. But the enzymetic activity was inhibited by 5 mM of CuCl₂, MnCl₂, Fe²⁺ or Fe³⁺.Under natural conditions, bacterial strains produce very low quantity of lipase. Direct utilization of lipase-producing bacteria is difficult to satisfy the need of industry. At present, people focus on cloning of lipase gene, and regulation and control of gene expression, in order to improve the production of lipase. Our study has offered a fast and effective method to obtain thermostable lipase, which shows an extensive prospect commercially. Currently, Thermus thermophilus HB27 has been a model strain for themophile research since the completion of the genome sequence in 2004. In this investigation, T. thermophilus WL was obtained from an offshore hot spring in Xiamen. Based on SDS-PAGE and Tricine-SDS-PAGE, the WL strain demonstrated two diffentent protein profiles at various growth temperatures. In order to obtain more differentially expressed proteins related to thermoadaptation, 2-D electrophoresis was performed with sample cultured at 55℃or 75℃. The 17 differentially expressed proteins as revealed by SD-PAGE, Tricine-SDS-PAGE and 2-D were subjected to mass spectrometry. Northern blots indicated that the 17 genes were up-regulated at higher temperatures, which were consistent with those of protein electrophoresis. Western blot confirmed the presences of SOD, FUH and TE-RF.SOD mutant was constructed with gene knockout. Surprisingly, the maximum temperature of SOD^- decreased from 80℃to 75℃. In order to confirm this result, the recombinant SOD was supplemented in culture medium. It was found that the SOD mutant recover growth at 80℃.Based on co-immunoprecipitation with SOD antibody, 2-oxoisovalerate dehydrogenaseαsubunit was found to interact with SOD. On the basis of validation with bacterial two-hybrid system, 2-oxoisovalerate dehydrogenaseαsubunit was subcloned into 3 segmental parts in order to look for the functional region. The results showed that 2-oxoisovalerate dehydrogenaseαsubunit could only have an effect as a whole molecule.Our studies revealed proteins related to thermoadaptation of thermophiles for the first time. These proteins were proteins involved in energy metabolism, proteins related to chromosome stability, thermal-stable proteins and SOD.