Recombinant Expression And Application Of Alkaline Protease From Vibrio Sp.DA1-1

Proteases are important in industrial production and in the study of life science.The study of its diversity is of great significance to reveal the process and nature of life activities.Degradation of specific proteins by protease can prepare peptide compounds with physiological functions,and can also be used in the preparation of biological materials containing protein components.However,specific degradation of the peptide bonds is the key to obtain active peptides or biological materials,and specific proteases need to be screened.At present,high-quality protease gene resources are limited,and it has been reported that the expression level of enzyme species is generally low,which affects the industrialization research and application to a certain extent.In this paper,an alkaline protease gene AprV from the Vibrio sp.DA1-1 was studied.The restructuring expression,purification and enzymology properties research work was done.Its hydrolysis of sea cucumber protein and active polypeptide preparation was studied.The main research results are as follows:?1?An alkline gene of AprV was cloned from Vibrio sp.DA1-1 with the size of 1200 bp.The recombination expression plasmid was successfully constructed with pET28a,and named as pET28a-aprV.The plasmid pET28a-apr V was transformed into BL21?DE3?p LysS.The protease activity of the recombinant strain was 45.81 U/m L.The sequence alignment was performed in NCBI.Serine protease from Alkalimonas collagenimarina was found to have the highest similarity of 68%with AprV protease.The AprV protease,which belonged to the peptidase S8 family,had the active site of Asp?169?,His?202?,and Ser?358?.A homologous modeling of AprV was carried out using the highest homologous protein crystal structure in PDB database as the template.The protein was formed by 11?-helix,11?-sheet and some random curls.?2?The optimum induction conditions of the recombinant Escherichia coli BL21?DE3?p LysS?pET28a-aprV?were analyzed,and the fermentation activity of AprV protease was increased by 1.9 times.The total activity reached up to 90.58 U/mL.The 6×His tag was introduced to the AprV protease,and the protease was purified by using Ni-NTA and Superdex 75 10/300 GL column.The recombinant protein was purified to electrophoretic homogeneity and the actual molecular mass was about 28 kDa.The recovery of purification was 8.98%and the purfication fold was 25.09.The specific activity of purified protein was1139 U/mg.The AprV protease contained a N-terminal propeptide and a C-terminal catalytic domain,and the N-terminal propeptide was automatically removed during the formation of the mature protein.?3?The enzymatic properties of the purified protease were investigated.The results showed that the optimum temperature and p H of AprV were 55°C and 10.0,respectively.The enzyme has good stability in an abroad range of pH 7.0-12.0.The protease activity retained more than 70%of activity after incubating at 40°C for 40 minutes,while only less than 40%of activity was remained after incubating at 50°C for 20 minutes.AprV protease belongs to metalloproteinase as its activity was completely inhibited by 20 mM EDTA.Protease AprV showed favorable tolerance towards Tween 20-80,JFC-2,H₂O₂,and DTT except for SDS.2mM K⁺,Na⁺,Ca²⁺,Mg²⁺,Zn²⁺,Ba²⁺and Co²⁺had slight activation on AprV protease,while 2mM Cu²⁺,Mn²⁺enhance the activity of AprV.The activity of Apr V was obviously inhibited by 5 mM Sn²⁺,with the relative activity of 33.6%.Through directed evolutionary techniques,a mutant M5 was successfully obtained with 1.3-fold enzyme activity increased.?4?In order to prepare the BAP of the sea cucumber,AprV protease was used to degrade the sea cucumber.Compared to commercial proteases,the present protease showed prefered application performance in preparing BAP of the sea cucumber,which had a higher protein content.SDS-PAGE analysis observed several proteins and peptides with small molecular weight and the degree of hydrolysis was 16.7%.The obtained sea cucumber polypeptide has a molecular weight of 100-1500 Da.Meanwhile,AprV protease can be used for the preparation of collagen peptide.Collagen peptides with molecular weight between 100-1500 Da were successfully obtained.