Study On The Characteristics Of A Marine Bacteria Producing Collagenase And The Biochemical Characteristics Of Collagenase

Compared with animal and plant-derived collagenase,microbial-derived collagenase has many advantages such as easy availability and short incubation time,and is widely used in various industries such as food,medicine and new energy.Therefore,the development of high-quality microbial source collagenase producing bacteria,increasing the enzyme production of the strain and studying the properties of the collagenase produced are of great significance for promoting the application of collagenase and the development of enzyme preparations.The main experiments and results of this study are as follows:(1)In this study,fish skin hydrolysis test and enzyme activity comparison test were used to select strain AL-13 as the experimental strain from 8 strains known to produce collagenase.The strain AL-13 was identified and constructed the phylogenetic tree by physiological and biochemical characteristics and 16S r DNA gene sequence analysis.The results showed that the strain AL-13 was Brevibacillus laterosporus.(2)Then we optimized the production conditions of collagenase by strain AL-13.With the activity of collagenase as the index,we used single factor experiment to optimize the culture conditions such as incubation time,incubation temperature and so on.The carbon and nitrogen sources of enzyme-producing medium were determined by single factor experiment,and the enzyme-producing medium was optimized by response surface methodology.Through optimization of enzyme production conditions,it was found that strain AL-13obtained the maximum enzyme activity(154.89 U/m L)at 25?,initial p H 8.0,inoculation volume 6%and 48 h of fermentation culture.At this time,the enzyme production medium was 8.14 g/L of glucose,11.63 g/L of beef extract,0.17 g/L of calcium chloride,2.08 g/L of potassium hydrogen phosphate and 9.48 g/L of sodium chloride.Compared with the enzyme activity before optimization(16.14 U/ml),the maximum enzyme activity after optimization increased by about 9.6 times.(3)Most of the impurity proteins in the fermentation broth of strain AL-13 were removed by ammonium sulfate fractional precipitation,and the purified crude collagenase was obtained.The specific activity of the purified crude collagenase increased from 10.02U/mg to 22.11 U/mg,the purified multiple was 2.21 times,and the recovery rate was 80.04%.Then we studied the enzymatic properties of collagenase such as temperature,p H and kinetic properties when casein was used as substrate.The results showed that the optimum temperature of enzymatic reaction was 50?,the optimum p H value was 7,the optimum metal ion was Mn²⁺,the Km value of tyrosine from casein hydrolyzed by collagenase was2.86 mmol/L,the Vmax value was 136.99 mmol/(L·min),and the gel was prepared.Proprotease is not resistant to high temperature or strong acid or alkali.It can be preserved at20-30?and p H 5.5-7.5,with the least loss of enzyme activity.(4)The hydrolysis of terramycin bacterial residue by collagenase from strain AL-13was studied to decomposition of mycelial cells,reduce the cost and difficulty of treatment of bacterial residue,improve the utilization of terramycin bacterial residue.The results showed that the hydrolysis rate was the highest(44.03%)when the adding time was 48 h,the hydrolysis time was 60 h and the adding amount was 15%.The protein content of terramycin bacterial residue was determined by taking 15 g of initial terramycin bacterial residue as an example.Before hydrolysis,the total protein content of the terramycin bacterial residue was7.86 g.After hydrolysis,the total protein content of the terramycin bacterial residue was 4.13g.The total protein content of the hydrolyzed terramycin bacterial residue was reduced by45.12%compared with the unhydrolyzed terramycin bacterial residue,and the result was similar to the reduction of the terramycin bacterial residue quality(hydrolysis rate).