Screening Of The Bacterial Strain Producing High Level Of Alkaline Protease And Study On Its Enzymatic Properties

Alkaline protease is a class of protein hydrolases with catalytic activity in the neutral to alkaline p H range.They have been widely applied in the food,pharmaceutical,detergent and leather manufacturing industries.Microorganisms are the main source of alkaline proteases.The aim of this study was to screen a microbe for high alkaline protease production from natural environments and characterize the properties of the produced alkaline protease.Here,an alkaline protease-producing strain was obtained from different environment and identified.Then,the fermentation conditions for producing alkaline protease were optimized using a single factor experiment,the enzyme was sequentially purified by ammonium sulfate precipitation,Sephadex gel chromatography and ion exchange chromatography.The effects of organic solvents,temperature,H₂O₂,p H,surfactants and metal ions on the enzymatic activity of the alkaline protease were investigated.And the optimum substrate and kinetic constants of the enzyme were determined.Finally,the encoding gene was cloned,and the main factors affecting the catalytic activity of the enzyme were analyzed using molecular docking and dynamics simulation to obtain the conformational changes of the overall structure of the enzyme,especially the region near the catalytic active site.The main results of the study are as follows:（1）Screening and identification of alkaline protease-producing strain:An alkaline protease-producing strain was screened from the compost soil sample by using skim milk agar medium and enzyme activity assay.Single colonies were obtained by plate scribing separation.Based on morphological,physiological and biochemical analysis,and 16S r DNA sequencing,it was identified and named as Bacillus atrophaeus L1.（2）Optimization of fermentation conditions for alkaline protease production by Strain L1:The fermentation conditions for alkaline protease production by B.atrophaeus L1 were optimized using a single factor experiment.The optimal fermentation medium for enzyme production was xylose 2.0%,peptone 2.0%,Na₂HPO₄.12H₂O 1.5%,KH₂PO₄ 0.3%,Mg SO₄.7H₂O 0.05%,and Ca Cl₂ 0.1%.The optimal fermentation conditions were cultivation temperature of 37℃,rotational speed of 180 r/min,250 m L flasks with a loading volume of 50m L,inoculation volume of 4%,initial fermentation p H of 6.0,and fermentation time of 30 h.After optimization,the enzyme activity can reach at 8941.46 U/m L,which is 12.36 times higher than the initial one.（3）Isolation and purification of alkaline protease:The alkaline protease produced by B.atrophaeus L1 was preliminarily separated and purified by ammonium sulfate precipitation.After desalting by buffer dialysis,it was purified by Sephadex G-75 chromatography column and cation exchange chromatography,and the electrophoresis pure protease was obtained.The recovery rate was 1.85%,and the purification multiple was 25.44.It was found that the relative molecular weight of the purified alkaline protease was about 28 k Da by SDS-polyacrylamide gel electrophoresis,and it was named as APL1.（4）Study on the enzymatic properties of alkaline protease:The results showed that APL1has the best catalytic ability at 60℃and p H 9.0,respectively.APL1 can keep stable in the range of 30～40℃and p H 7.0～10.0.The activity of APL1 can be increased to 137%by adding 10mmol/L of Mn²⁺,while Cu²⁺,Fe³⁺,and Al³⁺have significant inhibitory effect on its activity.Among various inhibitors,PMSF has the most significant inhibitory effect on enzyme activity,indicating the presence of serine residues in the active center of APL1.APL1 maintained good activity in 10%of various organic solvents,while it can still maintain relative enzyme activity of over 85%in 30%of ethylene glycol,glycerol,and DMSO.APL1 also exhibits good tolerance to non-ionic surfactants,the activity can be increased to 144%by Triton X-100 at the concentration of 10%.After treating with 1%and 5%H₂O₂ for 1 hour,APL1 retained 56.84%and 26.63%of enzyme activity,respectively.The substrate specificity experiment proved that APL1 had the highest hydrolysis efficiency for casein（100%）.While the degradation ability of skim milk and ovalbumin were 38%and 21%,respectively.The kinetic constants of APL1 were measured with casein as substrate,and the K_m value and V_max value were 13.9 mg·m L^-1 and181.8 mg·m L^-1·min^-1,respectively.（5）Gene cloning of APL1 and mechanism analysis of factors affecting enzyme activity:Primers were designed based on the homologous genes of alkaline proteases from B.atrophicus found in the NCBI database.Based on the results of protein mass spectrometry identification,the coding gene for APL1 was cloned from B.atrophaeus L1 using PCR,and the result showed that APL1 has a total length of 1092 bp and encodes 363 amino acids.Then,molecular dynamics simulations were used to calculate and analyze the main factors affecting enzyme catalytic activity.The structural changes of APL1 as a whole and enzyme catalytic activity centers before and after the addition of different factors were obtained through molecular docking,and the"structure-activity relationship"between different factors and enzyme catalytic activity were systematically studied.We found that H₂O₂,surfactants and organic solvents affect the activity of enzymes by covering or binding to key amino acids at their active centers through hydrogen bonding or hydrophobic interactions,while metal ions influence the structure of enzymes based on their polarity,directly leading to changes in enzyme catalytic activity.In summary,a highly alkaline protease producing strain B.atrophaeus L1 was screened in this study.The optimum fermentation conditions were obtained by single factor optimization experiment.Through the study of enzymatic properties,it was found that APL1 has good stability at 30～40℃and p H 7.0～10.0,and has a certain tolerance to H₂O₂,organic solvents,and surfactants.Mn²⁺has a significant promoting effect on its enzymatic activity,which indicated that it has certain application potential in detergents and others.The mechanism of different factors affecting enzymatic properties was further analyzed by molecular docking and dynamics simulations,which provides a certain basis for later research on the structure,function,and application of this enzyme.