Study On Producing Strains Isolation,Fermentation,Purification And Characterization Of Serratiopeptidase

Serratiopeptidase is a proteolytic enzyme,which possesses fibrinolytic,anti-inflammatory and anti-edemic?prevents swelling and fluid retention?activity in some tissues.Its anti-inflammatory effects are superior to other proteolytic enzymes.Presently,the production technique of serratiopeptidaseis is blank in China,so that developing a production process for serratiopeptidase with high yield is urgently needed.For this purpose,Isolation of serratiopeptidase producing strains,optimization of fermentation conditions,purification of the enzyme and enzymatic characteristics were studie in the paper.Firstly,seven strains,which can grow well on milk agar with red clones at 30?,were isolated from the intestines of silkworms collected from Lin'an,Hu'zhou?Zhejiang Province?and Guangzhou?Guangdong Province?.A strain designated as LL-413 exhibited excellent proteolytic activity when grew on the milk agar at 30?,and has red colonies with a transparent circle around clones.The activity of serratiopeptidase from strain LL-413 was 113.4 U/mL.By morphological identification and sequence analysis of 16S rDNA,LL-413 was identified as a strain of Serratia marcescens.Secondly,for increasing the yield of serratiopeptidase,the single factor experiment and response surface methodology were applied to optimize the enzyme production process.The results showed that the optimal process was as follows:The medium composition for enzyme production are 11 g/L yeast extract,6.6 g/L beef extract,12.2 g/L malt extract,1 g/L MgSO₄ and 5 g/L NaCl with the initial pH 8.0;the fermentation temperature is 30?,and the fermentation time is 40 h.Under the optimized conditions,the activity of serratiopeptidase was achieved at 1126 U/mL,increased almost ten times to the activity of113.2 U/mL before optimization.Thirdly,a pilot experiment was carried out by 10 L bioreactor.When loading volume of medium was 6.5 L,fermentation pH was controlled between 7.5⁸.5,oxygen saturation was maintained higher than 50%,cultural temperature was set at 30?,and fermentation time was 8 h,the enzyme activity was achieved at 1534 U/mL with the biomass of 9.8 g/L,improved 36.2%compared with that obtained under flask cultivation.Finally,the separation process for the serratiopeptidase purification from the strain LL-413 was developed,and enzymological characteristics of serratiopeptidase were studied preliminary.By steps of salt precipitation,dialysis,ultrafiltration and DEAE-Sefinose ion exchange chromatography successively,the activity of serratiopeptidase was achieved at 8044 U/mg,increased about 2.1 times.The recovery of enzyme activity was 20.7%.Using the purified serratiopeptidase,the enzymological characteristics was investigated.The results showed that the optimum temperature and pH of serratiopeptidase activity were 40?and 8.0 respectively.The kinetic constants K_m and V_maxax were 22.84 g/L and 1.37 U/min respectively.The fibrinolytic activity was about 100000U/g.The molecular weight of serratiopeptidase from the strain LL-413was estimated about 50 kDa by SDS-PAGE.