Cloning Of Protease From Planococcus Bacterium And Its Application In Preparation Of Antioxidant Enzymatic Hydrolysates

Psychrophilic/cold-adaptive microorganisms are a type of microorganisms that have evolved to adapt to cold environments,and generally distributed in low temperature environments such as polar regions,deep seas and high altitudes.The cold-adapted enzyme produced by these psychrophilic/cold-adaptive microorganisms own characteristics of high catalytic efficiencies at low temperature and flexible structure,and have great potential for innovation in the food and molecular biology industries.In this paper,the genomic DNA of psychrotrophic bacterium Planococcus maritimus XJ11was sequenced,and its protease genes were cloned and expressed.Moreover,the recombinant protease was utilized for proteolytic hydrolysis of silver carp fish meat to prepare antioxidant products.The main research results are as follows:(1)De Novo whole genome sequencing was performed on P.maritimus XJ11.The results of genome sequencing showed that P.maritimus XJ11 possessed a 3282604bp circular chromosome and a 67339 bp circular plasmid with a total of 3293 open reading frames(ORFs).According to the annotation results of the NR database,a total of 21 protease coding genes,including 3 serine protease,were identified.Moreover,five protease genes(xj11-288,xj11-386,xj11-467,xj11-2299 and xj11-2529)were selected for basic bioinformatics analysis,such as physical and chemical information,hydrophilicity and hydrophobicity,signal peptide and transmembrane structure.(2)Engineered E.coli bacteria were constructed to express five protease genes(xj11-288,xj11-386,xj11-467,xj11-2299 and xj11-2529).The results showed that the heterogeneous expression of xj11-288,xj11-467 and xj11-2529 were successfully completed,but none expression products of xj11-386 and xj11-2299 were detected.Among the PmSpr-288,PmCpr-467 and PmSpr-2529 products encoded by xj11-288,xj11-467 and xj11-2529,only PmSpr-288 exhibited proteolytic activity,and its optimal catalytic temperature and p H were 35°C and 11.0,respectively.Furthermore,it showed good stability to medium and low temperature and good tolerance to alkaline p H.Divalent metal ions Mg²⁺,Ca²⁺,Co²⁺,Cu²⁺had slightly stimulation effect on the activity of PmSpr-288.However,10 m M Fe³⁺and Mn²⁺significantly inhibited the activity of PmSpr-288.EDTA and PMSF completely inhibited the activity of PmSpr-288.Nevertheless,PmSpr-288 had a certain tolerance to ethanol,isopropanol and acetonitrile.When casein was used as the substrate,the kinetic parameters K_m and V_maxvalues were 14.61 mg/m L,3.104×10³?g/min/mg,respectively.Bioinformatics analysis showed that PmSpr-288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49,His86 and Ser251.In addition,PmSpr-288 was co-expressed with five chaperone plasmids(p G-KJE8,p Gro7,p KJE7,p G-TF2 and p Tf16).The results showed that the soluble expression and enzyme activity of PmSpr-288 were increased in different degrees.(3)PmSpr-288 displayed good hydrolytic ability to both silver carp myofibrillar protein and myosin.The amino acid nitrogen content of the enzymatic hydrolysates was158±3.61?g/m L and 194.83±10.69?g/m L,respectively.In addition,RSM tests were carried out to optimize the properation process of anti-oxidant enzymatic hydrolysates.The optimal conditions were set as follows:enzyme additive amount is 4855.47 U,solid-liquid ratio is 10.09,and enzymatic hydrolysis time is 1.0 h.The enzymatic hydrolysis product obtained under these conditions showed a DPPH free radical scavenging rate of 66.34%,and presented an IC₅₀ value of 1.309 mg/mL.