Isolation And Identification Of Protease-producing Bacillus And Its Crude Enzyme Properties

Researchers are concerned about moderately adaptable moderately halophilic bacteria because they can survive in a wide range of Na Cl content and are not very demanding on nutrients.Generally speaking,proteases secreted by microorganisms are extracellular in nature,so compared with proteases obtained from other sources,the downstream processing of enzymes is simplified.Many factors can affect the performance of proteases,such as p H value,ionic strength,heat and so on.Due to the huge demand for proteases in the industry,researchers are constantly exploring different aspects of proteases.The purpose of this paper is to provide new strains and enzyme reagents for industrial production and application.Many researchers agree that biological treatment of high-salinity sewage is technically feasible because the halophilic bacteria catalyze the hydrolysis of pollutants under high-salinity conditions.The use of halophilic microorganisms to act on high-salinity wastewater has a bright future in the fields of improving the harm caused by wastewater and high-salinity wastewater to the environment because of its low cost and quick results.Microorganisms have the characteristics of fast growth and reproduction,many types,easy cultivation,and large-scale release and extraction of proteases.Therefore,the screening and application of proteases secreted by microorganisms is a very important work in the hydrolysis and transformation of microorganisms.Each strain has its own unique physical and chemical conditions and nutrient requirements to maximize the synthesis of proteases.There is no definite common medium for proteases secreted by different microorganisms,and each microorganism or strain has its own maximum enzyme production requirements,which is conducive to the most suitable growth conditions will result in the highest enzyme production is not always correct.Various researchers have reported that the culture conditions that promote the production of proteases are different from those that promote cell growth.The research method used in this thesis is to isolate the strains by screening the solid medium with strains;to observe the morphology of the strains with the naked eye and scanning electron microscope and to analyze and compare the 16 S r DNA sequence resultsto identify the strains;to isolate the strains by their growth The strains are classified;the protease activity is measured by the Folin method;the influence of heat,p H,and Na Cl concentration on the protease activity is measured by the control variable method;the optimal fermentation time and the best nitrogen of this strain are found by the control variable method The source,the best carbon source,the best Na Cl concentration of fermentation and the effect of metal ions on enzyme activity,so as to determine the composition of the fermentation medium of this strain.The main research results of this article are as follows:(1)In this experiment,a strain that secretes both protease and halophilia was selected from the Shuangfeng Saltworks in Zhoushan,Zhejiang Province.The morphology of the rods was observed under a scanning electron microscope,and the results of 16 S r DNA complete sequence and strain morphology were confirmed as Halobacillus trueperi,and then named B1(Accession No.: MK367780).After research,the strain has an optimal Na Cl concentration of 5% and can survive at a salt content of 20%,but B1 is difficult to grow at a Na Cl concentration of 0%,indicating that it is a moderately halophilic bacterium.(2)The enzymatic properties of the protease secreted by the bacteria were investigated.The protease secreted by B1 bacteria was measured to be a medium-temperature protease because the best heat of its vigor was 50 degrees Celsius.It is a neutral protease because its highest protease activity is at p H 7.5.The activity of protease produced by strain B1 will be inhibited to some extent with the increase of Na Cl concentration.After the Na Cl content reaches 8%,the remaining vigor reaches 61.0%,which has good salt tolerance.(3)The protease activity of strain B1 reached its maximum value after 8 hours of fermentation.The optimization conclusion of the culture liquid nitrogen source reveals that skimmed milk powder is the best nitrogen source.Glucose,maltose,sucrose and fructose have significant inhibitory effects.If the salt content in the culture solution increases,the activity of the secreted protease will increase first and then decrease,and the enzyme activity will reach its maximum at the concentration of 7.5% Na Cl.The effect of Cu2 +,Zn2+,and Ni2 +ions on the activity of protease produced by strain B1 was significantly inhibited.