Analysis Of Culturable Microorganisms Diversity In Soda Lake,Taxonomic Analysis Of Novel Bacteria And Characterization Of The Protease Properties

Inner Mongolia Autonomous Region of China is located in the middle and high latitudes.The climate is dry,windy and rainless.The topography and climate conditions are conducive to the formation of alkali lakes.The high salinity environment of alkali lakes provides ideal conditions for the growth of haloalkaliphilic bacteria.In recent years,more and more attention has been paid to the research of haloalkaliphilic bacteria in the world,because the research in this field can not only enrich people’s understanding of microbial population diversity,but also find some novel halophilic and alkalophilic enzymes to meet the needs of modern industrial development for special enzymes.Therefore,in this paper,the haloalkaliphilic microorganisms in Inner Mongolia alkali lake water samples were screened,and two novel haloalkaliphilic bacteria were identified.On this basis,two saline-alkali resistant proteases from the two novel strains were isolated and characterized.Firstly,24 haloalkaliphilic bacteria were isolated from alkali lake water samples in Inner Mongolia by traditional enrichment culture method.The results of 16S r RNA gene sequence showed that 13 strains belonged to Halomonas,6 strains belonged to Alkalimonas,3 strains belonged to Salinispirillum,and 1 strain belonged to Roseinantronobacter and 1 strain belonged to Aliidomarina.In addition,the diversity analysis of 24 strains showed that 13 strains could produce protease and cellulase at the same time,8 strains could produce amylase,and 3 strains could produce lipase.Secondly,haloalkaliphilic strains LH10-3-1 and LH10-5-2 were identified by the multiphase taxonomic method.The results showed that strains LH10-3-1 and LH10-5-2 were gram-negative,spiral,flagellate,quinone type was ubiquinone Q-9,and the main fatty acids were Summed Feature 8 and C_16:0,which accorded with the main characteristics of genus Salinispirillum.The ANI and GGDC values between strains LH10-3-1 and LH10-5-2 and the reference type strain Salinispirillum marinum GCWY1^T were 73.32%and 74.42%,27.10%and 27.20%,respectively.The two strains were identified as different strains of the genus Salinispirillum,named Salinispirillum marinum sp.nov.LH10-3-1 and Salinispirillum marinum sp.LH10-5-2,respectively.The molecular weights of protease AP-1 and AP-2 are 38 k Da and 45.7 k Da,the optimal reaction conditions are 50°C,p H 9.0 and 60°C,p H 9.0.Ca²⁺,Mg²⁺,and Cu²⁺are activity promoters,while reducing DTT andβ-mercaptoethanol are strong inhibitors for both enzymes.The metal chelator EDTA promotes the activity of protease AP-1,but has no effect on protease AP-2.When casein was used as the substrate,the K_m and V_max values of protease AP-1 and AP-2 were 0.6285 g/m L,1.257 mmol.L^-1.min^-1 and0.5472 g/m L,1.095 mmol L^-1.min^-1,respectively.Finally,the protease AP-1 and AP-2 produced by strain LH10-3-1 and LH10-5-2were extracted and characterized by ammonium sulfate precipitation and gel filtration chromatography.The results showed that the specific enzyme activities of AP-1 and AP-2 were 2632.0 U/mg and 3639.0 U/mg respectively,and the purification folds were15.3 times and 13.9 times,respectively.The molecular mass of protease AP-1 and AP-2 was 38 k Da and 45.7 k Da,respectively.The optimum reaction conditions were 50°C,p H 9.0 and 60°C,p H 9.0.Ca²⁺,Mg²⁺,and Cu²⁺were active accelerators,while reducers DTT andβ-mercaptoethanol were strong inhibitors.EDTA promoted the activity of protease AP-1,but had no effect on the protease AP-2.When casein was used as substrate,the K_m and V_max values of AP-1 and AP-2 were 0.6285 g/m L,1.257 mmol L^-1 min^-1 and 0.5472 g/m L,1.095 mmol L^-1 min^-1,respectively.