Cloning And Expression Ofaspergillus Oryzae Gif-10 Alkaline Protease Genes Alp1 And Alp2 In Pichia Pastoris

Alkaline protease is the enzymes that the reaction optimum pH is in the alkaline-range.Research shows that most of the alkaline protease is serine protease. A small amount of alkaline protease is metalloproteinases.Alkaline protease has been widely used in detergent, leather, silk, medicine, food, feed, environmental protection, waste disposal, chemical industry and in the field of basic research.There are statistics show that alkaline protease sales accounts for 25% of all sales of industrial enzymes. The alkaline protease from microorganisms are mostly extracellular enzyme,it’s suitable for industrial production,and can be widely used because of it can be simple cultivated,cast low,can be high yielded etcAspergillus oryzae is the strain that can produce many kinds of enzymes.lt can produce proteases,amylases,cellulases,phytases etc.Aspergillus oryzae is widely used in food, feed, production of kojic acid, wine and other fermentation industry.It is safely used for over 1000 years. Biochemistry laboratory isolated the Aspergillus oryzae gif-10 from soil humus layer. The strain have be identified with strong alkaline protease activity. This study intends to clone alkaline protease gene from aspergillus oryzae gif-10 and efficiently express in Pichia pastoris.Using the amino acid sequence of published alkaline protease KerA BLAST with aspergillus oryzae RIB40 genome database.Alkaline protease alpl and alp2 got the highest score. Design primers and PCR clone aspergillus oryzae alpl and alp2’s gene sequence into vector pMD19TAspergillus oryzae’s total RNA was isolated and reverse transcripted into cDNA. Alpl and alp2 CDS seuqence were PCR cloned into pMD19T. After sequencing, we find that the alpl CDS is 1212bp long, coding a protein of 403aa, it’s gene is 1377bp long and have four exons.The alp2 CDS is 1488bp and have a ORF coding a protein of 495aa,it’s gene is 1610bp and have three exons. The amino acid sequence of alkaline protease alpl and alp2 were aligned at NCBI. The identities is 41%. Alp2’s carboxyl terminus have a 44aa sequence that contain a high a helix sequence of unknown function.Design primers to amplify alpl and alp2 cDNA sequence that without signal coding sequence. The sequence was digested with EcoRl and NoiI before clone into vetor pPICZaA. I have successfully constructed the pichia expression vector of pPICZaA-alpl pPICZaA-alp2 and pPICZaA-△alp2(delet C end 44aa). They were linearized with SacI and electroporated into Pichia pastoris X33.After the Zeocin resistance screening and identification by PCR, I obtained several positive clones for each of the expression vector. After induced by carbinol i have screened three pichia engineering strain with highest active:X33-alpl-3, X33-alp2-2 and X33-△alp2-2. At the 5th day of induction, pichia engineering strain X33-alpl-3 get it’s highest enzyme production of 36.9U/mL.It’s specific activity is 66.9U/mg. The highest secretion level of stain X33-alp2-2is 2.4U/mL. The highest secretion level of stain X33-△alp2-2 is 40.5U/mL.It’s specific activity is 70.1U/mg. The pichia engineering strain X33-alpl-3 and X33-△alp2-2 were induced by carbinol. After the crude enzyme solutions were concentrated by ultrafiltration,SDS-PAGE analysis and enzymology properties research was taken. The results showed that, the recombinant alkaline protease alpl and alp2 have molecular weight of about 29kD and 32kD. The optimum reaction pH of recombinant alkaline protease alpl is 10,then △alp2 is 8.5. After store at 50"C for 2 hours the residual enzyme activity is 83% and 63% for recombinant alkaline protease alpl and △alp2 respectively.The optimal reaction temperature is 65℃ and 60℃ for alp1 and △alp2 respectively when the reaction time is 10 mins.Alpl and △ alp2 has very strong ability to degrade casein and gelatin,they can degrade beef powder,but they have a slight degradation of feather keratin and keratin azure. CaCl2、CoCl2 and MgCl2 can improve the active of recombinant alkaline protease alpl and △alp2. Zn2SO4、CuSO4. FeSO4 will inhibit the active of recombinant alkaline protease alpl and Aalp2. PMSF can completely inhibite the active of alp1 and △alp2. EDTA have significant effect on the activity of alp1 and △alp2.