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Preliminary Research on Characterization of Thermophilic Protease Pasey and Helicase Htc16

Author: LiaoYanJiang
Tutor: LinLianBing
School: Kunming University of Science and Technology
Course: Microbiology
Keywords: Thermophilic Protease Purification Characterization Helicase Bioinformation Analysis
CLC: Q814
Type: Master's thesis
Year: 2011
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Abstract


Thermostable enzymes are widely used in industries for their high catalytic reaction and high thermal stability. A thermostable extracellular protease producing strain GSEY01 was isolated from a hot spring in Rehai, Tengchong, Yunnan Province. The strains in the solid medium 2% casein agar can produce clear proteolytic laps, its hydrolysis circle diameter/colony diameter is 0.6. The optimal growth temperature of strain GSEY01 was 60℃.16S rRNA gene phylogenetic analysis showed that this strain belonged to thermophilic Geobacillus. sp. Protease secreted by strain GSEY01 was purified to homogeneity by ultra filtration, ammonium sulfate precipitation and ion exchange chromatography. Molecular weight of this proteinase was 42 kDa. The protease showed the highest activity at 80℃and pH 7.5. The activity of the protease could be activated by Mg2+, but inhibited partially by Fe3+、Cd2+and Ni2+. In addition, the activity of the enzyme was inhibited by EDTA and SDS strongly, but not inhibited by PMSF. Kept in 98℃for 30min, Pgsey had 17 percent residual activity. Protease from strain GSEY01 was greatly different from those proteinase isolated from other strains of Geobacillus, and may be applied in industrial processes that are performed under high temperature.Helicases are motor proteins that use the free energy of NTP hydrolysis to catalyze the unwinding of duplex nucleic acids. Helicases participate in almost all processes involving nucleic acids. Their action are critical for replication, recombination, repair, transcription, translation, splicing, mRNA editing, chromatin remodeling, transport, and degradation.In this study, helicase gene primers were designed according to the helicase conservative sequence of Thermus thermophilus, and helicase gene htc16 from Thermus strain TC16 were amplified and sequenced. htc16 coded 444 amino acids. The protein Htcl6 sequence similarity of that of Thermus strains were 84%. Helicase conservative domains analysis showed that Htcl6 belonged to DnaB super family and contained two motifs:walker A motif and Walker B motif. Homologous modeling analysis indicated the protein structure of Htcl6 was similar to the model of 2q6aA. Gene htc16 was cloned to vector pET-28a to construct an expression plasmid pET28a-htc16, and the plasmid was introduced into BL21 (DE3) cells to obtain an express strain BL21/pET28a-htc16. Htc16 recombinant protein was expressed insolubly in E.coli BL21 strain after induced by 1mM IPTG for 8h. The recombinant protein molecular weight was about 53.8 kDa.

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