Characterization Of Protease From Strain Asp-195v Of Aspergillus Clavatus And Its Partial CDNA Gene Cloning

Protein hydrolytic enzymes make the peptide chain break in the protein, and they are important groups of enzymes both in physiology and commerce fields. Protein enzymes execute massive different physiology functions, including the normal and abnormal conditions which are responsible to the complex pathology, physiological function. They also exist in the pathogenesis organism's life, so they can be used to cure some terrible disease, such as cancer and HIV. There is a glorious history about the proteases in many fields, for example: food, fur and leather industry, medical and the cleanser industry. The protease coming from microorganism, because of its easily culture, rich output, and suitable for the industrialization production, can be used widely.In the first period of this paper, the optimal condition of producing-protease by Asp-195v was studied; the fermenting liquor from Asp-195v was purified to obtain a single protein component, and then the characterization was studied. The results are as follows.(1) The optimum conditions were as follows: the bran and bean cake in the medium content was 7:3,the liquid level was 90mL, cultivating solution initial pH was 7, incubation time was 72h, the inoculation size was 1.5mL, the incubation temperature was 24℃in 250mL triangle bottle, rotation speed was 120 r/min. Under the best circumstance the protease activity was 309.70U.(2) One kind of proteases was purified from the Asp-195v fermenting liquor by the (NH4)2SO4 deposition, Sephadex G-100 gel exclusion chromatography and DEAE FF Sepharose these three steps. And the molecular weight is 30.1kDa, the sample has been purified 15.3 times, the yield is 6.35.(3) The test of the enzyme characteristics shows that the optimum temperature for its activity was 40℃, and its activity remained more than 70% from 30℃to 50℃. Its activity remained stable in the range of pH 4-9 with the optimum requirement of pH 7. Mn²⁺ had an obvious activation on the protease activity, and its activity was obviously inhibited by K+, Ag⁺, Cu²⁺, Fe²⁺, Mg²⁺, Zn²⁺, Ca²⁺, Al³⁺ and Fe³⁺, however, it was severely inhibited by Hg²⁺ and Pb²⁺. Other reagents such as glucose and EDTA make little inhibition to the enzyme, on the contrary the sucrose, SDS and Tween-20 do. Its Vmax and Km were 30.40mmol/min, 97.53mmol/L. The primer was designed by basing on the conserved amino acid sequences of the producing-protease fungi, rapid cloning gene from cDNA Library by PCR (517bp). The gene has been submitted to the GenBank with accession number GU455424. Then RACE will be performed to obtain the complete mRNA, so its function can be used for further study.