Screening Of A Marine Strain Producing Protease And Optimization Of Fermentation Conditions

China has vast ocean area and rich Marine resources. As an important Marinebiological resource, Marine microorganisms have more than1million species. However,less than5%of them are researched and identified currently. As a protein catalytichydrolysis of the enzyme, protease has become the most widely used and the greatestdemanded enzymes currently. Traditional protease drawn from animals and plants has thefollowing other shortcomings, such as long production cycle, high production costs,difficult management control, vulnerably restricted by land and seasons and so on.Microbial protease is increasingly becoming a major source of microbial industrialenzymes. Currently, the industrial production of protease is mostly from land or freshwater in our country.1.In view of the limited source of our domestic microbial proteases, single types ofprotease products, relatively expensive price, not enough in-depth and extensive industrialapplications, and the research, development and utilization of marine microbial resourcesstill far behind the developed countries and other issues, this study collects99samples ofseawater from the Bohai Sea. A strain capable of producing protease is first got by thescreening of flat transparent circle method and rescreening of Folin phenol reagentcolorimetric method. Then the strain is identified through morphological, physiologicaland biochemical characterization and16SrRNA sequence analysis. Finally, thecomposition of producing nutrient medium and enzyme fermentation conditions areoptimized through the response suface method, orthogonal experiment method, whichsignificantly improves the protease activity and lays foundation for the later strainimprovement and industrial production. The results shows that:2.Identification of strains. On casein plate diameter of5mm, strain22-1colonies areround, milky white, opaque, neat-edged, smooth-surfaced, short rod-shaped, Gram-positive,spore, and easy to picked up and produce a clear transparent circle. Moreover, it wasconsistent with the characteristics of Bacillus pumilus of the Bergey’s manual. After16SrRNA gene sequence compares with Blast, it turns out that the highest homology strainis Bacillus pumilus, whose homology is99according to the sequences provided inGen-Bank. Therefore, the strain was identified as Bacillus pumilus.3.Optimization of fermentation medium nutrients. After discussing the effect of carbon, nitrogen, and surfactants on the enzyme activity, this experiment uses three factorsand three levels of response surface methodology to explore the influence of corn flour,soybean meal and Na2HPO4on enzyme activity and forms a mathematical model betweenenzyme activity and various main factors based on single factor experiments. The multipleregression equation was:Y=219.27+0.29*A-4.29*B+8.64*C+1.07*A*B+13.87*A*C+17.28*B*C-20.50*A2-28.06*B2-23.82*C2，R2of the model was99.64%, then the equation derivation, extremecomputing, the optimal nutrient content:3.07%corn flour, soybean meal0.99%, Na2HPO40.84%. In order to verify accuracy and reliability of this model, three parallel fermentationtests are conducted in the medium under optimal conditions, and the average enzymeactivity of the three tests is218.87U/mL, whose relative error is less than1%comparingwith the theoretical predictions. Moreover, it improves6.14times over previousoptimization.4.Optimization of fermentation conditions. On the basis of single factor experiments,four factors and three levels orthogonal experiment is applied to explore the influence ofinoculum size, liquid volume, and temperature and fermentation time on the enzymeactivity. According to the test results based on a single factor and the variance resultsanalysis of poor SPSS software to orthogonal experimental results, it turns out that theimpact of fermentation time on the enzyme activity was quite observable (p<0.01), theeffect of training temperature on the enzyme activity was observable(p<0.05), the order ofthe effect of four factors: D(fermentation time)>C(incubation temperature)>B(inoculumsize)> A(medium volume), to determine the optimal combination:A2B2C2D2, namelyfermentation time72h, culture temperature37℃, inoculum size3%, loaded fluid volume70mL/250mL. Final determination in this fermentation broth under conditions of proteaseenzyme activity was267.53U/mL, optimization improves over the previous7.67times.