Study On Protease And Cloning Of Subtilisin Gene From Bacillus

One bacterial strain(sp. HX-12-5) producing high yield of neutralprotease with good dehairing effect,was ever isolated in our laboratory and the enzyme activity is 2665 u/mL. The strain was identified as Bacillus licheniformis.For B.licheniformis HX-12-5,the optimal fermentation conditions of yielding neutral protease were determined and the results are as follows. The composition of fermentation medium is corn flour 4 %,soy bean cake meal 3 %,bran 3 %,CaCO3 0.3 %,Na2HPO4 0.2 %,KH2PO4 0.2 % ,initial pH7.5. The optimal cultivation temperature is 34-37 ;time:48h;ratio of medium to container:1:5-1:7;speed:200rpm. Under these conditions,the neutral protease activity of the supernatant produced by the strain can reach 4500 u/mL or more.The study on the properties of crude neutral protease showed that the optimal reaction pH value and temperature are 7.0 and 50,respectively. The enzyme is stable at the range of pH 6-9 at 40. SDS,PMSF and EDTA partially inhibit the protease activity.A pair of primers,based on the high homologous N- and C- terminal amino acids sequences of subtilisin of Bacillus pumilus,were designed and used to amplify subtilisin gene from genomic DNA of 4 different Bacillus strains by polymerasechain reaction. As a result,a fragment about l.lkb was obtained from Bacillus sp.813 and cloned into T-vector and then sequenced. The result of sequencing shows that this fragment contains an open reading frame of 1106 nucleotides and encodes 369 amino acids residues,which is part of a alkaline serine protease,with 98.3% homogeneity to a reported gene from Bacillus pumilus. In the same method,another pair of primers weredesigned in order to amplify subtilisin Carlsbsrg from genomic DNA of those Bacillus strains by PCR. The cloning and sequencing of a fragment from Bacillus licheniformis DY. are under investigation.