Purification And Characterization Of An Alkaline Protease From Streptomyces Sp.,and Gene Cloning And Expression

Alkaline proteases?EC 3.4.21?have a number of special properties,such as stability under high temperature,alkaline agent,surfactant and organic reagents,etc..It is widely used in detergent industry,food industry,and leather industry.Compared with the alkaline proteases,commercial microbial proteases are significantly unstable.In addition,a majority of the industrial proteases are produced by Bacillus,alkaline protease from actinomycetes has been ignored.Therefore,screening resources from actinomycetes producing alkaline proteases has a very important significance for enriching protease resources and expanding practical application.In this study,actinomycetes producing alkaline protease were isolated from soil samples.Firstly,stains producing proteases were screened by transparent circle on Gause's agar medium containing casein,and then they were rescreened through the protease activity.Finally a strain with higher activity of alkaline protease was isolated,and its protease activity in the Gause's medium reached 138 U/mL.From the analysis by 16S rDNA sequence,the strain was Streptomyces sp.,and it was named as Streptomyces sp.M30.Various ingredients of fermentation medium and culture conditions were optimized in order to make the Streptomyces sp.M30 reached the maximum enzyme production.Optimized components of Gause's medium were:8 g/L soluble starch,1.7 g/L KC1,0.5 g/LKN03,0.5 g/L Na2HP04·12H20,0.5 g/L MgS04,0.02 g/L,CaC03,0.01 g/LFeS04.Culture conditions were:initial pH7.0,10%inoculum,the incubation temperature 20 ?.The activity after optimization up to 313 U/mL,and it was 2.3 times before optimization.And relative activity of SapH was increased by 127%.After fermentation,the properties of crude enzyme were studied.The optimum temperature of the crude enzyme was 80 ?,it was stable in temperature range of 0-50 ?and retained 85%of its initial activity after 2.5 h incubation at 60 ?.The optimum pH was 9.0,and the activity of SapH was stable over a pH range of 4.0-10.0.Furthermore,different metal ions have little effect on protease activity.The alkaline protease was named as SapH depending on the characteristic of the alkaline protease.Purification steps of alkaline protease SapH were:ammonium sulfate precipitation,hydrophobic interaction chromatography,and DEAE-sepharose chromatography,with a yield of 3.6%and a specific activity of 29071.8 U/mg,and concentrated the protein 4.7-fold.The molecular mass of SapH was 54.8 kDa based on MS.The Km and Vmax values were estimated to be 35.71 mg/mL and 5 × 104 U/mg,respectively.SapH exhibited highest activity at 80 ?,with relatively high activity over 70-85 ?.We also found that SapH was stable from 20-40 ?,and retained 65%of its initial activity after 2-h incubation at 50 ?.However,incubation at 60 ? for 30 min resulted in decrease of activity to 40%.SapH activity was observed over a pH range of 6.0-11.0,with maximum activity at pH 9.0.The activity of SapH was stable over a pH range of 4.0-10.0,with 90%activity between pH 6.0 and 10.0.SapH activity was significantly inhibited by EDTA,EGTA,DTT,and almost completely inactivated by the serine protease inhibitor PMSF,indicating that SapH was likely a serine-type protease.Protease activity was increased in the presence of Ni2+,Mn2+,and Cu2+ by 112%,113%,and 147%,respectively.In addition,enzyme activity was activated by DMF and DMSO,and was stable in the presence of methanol and acetone.The non-ionic surfactant Tween 80 enhanced enzyme activity by up to 121%.Purified SapH was subjected to peptide mass fingerprinting.Nucleotide sequence analysis revealed that the sapH gene contained 1,179 bp,corresponding to 392 amino acids.According to peptide mass fingerprint analysis,two internal amino acid sequences exhibited 100%identity with a hypothetical protein?WP₀19431870.1?from Streptomyces sp.AA0539.By constructing an expression vector,heterologous expression were carried out in E.coli expression system,Bacillus subtilis expression system and Pichia pastoris expression system.The results showed that expression vectors entered the host strain successful,but no protease activity was detected.