Screening Of Protease-Producing Marine Bacterias And Appling It On Fermenting Soybean Meal

With the refinement of using microbial strains and enzymes, the requirements for thestrains and enzymes applied to the industry have always been improved, especially theincreased demend for the strains and enzymes which is of high salt resistance and can stay inthe environment with a wide range of temperature and pH value, and also have some specialfeatures. In order to seek more resources of microorganisms and bioactive substances,researchers change their eye-sights into the ocean to find out the new strains and enzymes tomeet industrial requests. This study is about screening protease-producing marine bacterialstrains from samples of deep sea mud (the South China Sea), and optimizing the rockerfermentation condition for the screened strains before purifying and manufacturing theprotease. Moreover, the screened strain is used to ferment soybean meal to improve theanti-oxidation of the zymotic fluid. It is expected to find out a bacterial strain that has somespecial features or secrete protease which have some particular properties. The main resultsare as follows:(1)132protease-producing marine bacterial strains were isolated from the deep sea mudof the South China Sea. Among them, there were30strains exhibiting clear transparent circleon the casein-medium. Finally,2protease-producing strains, named as SWJS33and SWJS22,were chosen to further study due to their better properties of crude protease. These isolateswere identified and characterized as species of Bacillus licheniformis and Bacillusamyloliquefaciens respectively, based on their biochemical and physiological characteristicsand analysis of16S rDNA sequence.(2) Rocker fermentation conditions of the protease secreted by SWJS33and SWJS22were researched respectively. The optimum medium for SWJS33were maltose (0.5%), yeastextract (1.0%), calcium chloride (0.28%), sodium chloride (0.1%) and sucrose fatty acid ester(SE)-170(0.05%), and the optimum technics conditions of fermentation were initial pH ofculture medium was7.5, inoculums was1.5%, culture-broth-quantum was10%and culturetime and temperature were48h and37℃respectively. As for SWJS22, the optimummedium were lactose (0.5%), soybean meal (1.0%), potassium dihydrogen phosphate (0.05%),magnesium sulfate (0.03%), ammonium sulfate (0.1%), calcium chloride (0.1%) andglycerinum (0.05%). In the condition that initial pH of culture medium was9.0, inoculumswas1.0%, culture-broth-quantum was10%, and culture temperature and time were37℃and54h respectively, the strain SWJS22grew well and total protease activity reached its peak. As a result, the protease activity produced by SWJS33and SWJS22were6and9timesrespectively that produced by SWJS33and SWJS22before optimizing.(3) The protease secreted by SWJS22was purified through several steps, and the finalpurification-fold111.68and recovery rate21.17%were obtained. The molecular mass14.4KDa was estimated by SDS-PAGE. The optimal pH and the protease temperature were11and30℃, respectively. The purified protease had a strong tolerance on salt. There was alsoresidual enzyme activity of50.12%in the18%（W/V） sodium chloride solution. The enzymewas activated by Ca2+、Mn2+and Mg2+, and inhibited by Fe2+、Ni2+、Co2+、Cu2+and Hg2+. Zn2+had no effect on the protease activity. The enzyme was strongly inhibited byphenylmethylsulfonyl fluoride, and had almost no effect on EDTA, PCMB andβ-ME. Andthe BSA had a positive effect on protecting the enzyme when2μg/mL of BSA was mixedwith the enzyme. The enzymatic kinetic of purified protease was also studied, Kmand Vmaxvalues were determined to be3.690mg/mL and2.434mg mL-1min-1,respectively. And itsphysiological efficiency (Vmax/Km) was calculated to be0.6596min-1.(4) In order to study the antioxidant activity of fermented soybean meal which wasfermented by SWJS22, the DPPH free radical scavenging activity, reducing power andoxygen radical absorption capacity were measured to character the activity. The resultsshowed that the zymotic fluid had a strong antioxidant acticity when the fermentation timewas during54h to60h. And the antioxidant substances (MRIs, MRPs, TP, TF, and smallpeptides (<1KDa)) in the zymotic fluid soared during fermentation.