Activation And Enzymatic Property Of A Metalloproteinase(TvM6)from Thermoactinomyces Vulgaris CDF

Thermoactinomyces vulgaris CDF is a thermophilic strain isolated from the soil of Wuhan University.It can degrade intact feathers under high temperature and use the degradation products for growth,showing that this bacterium has certain application value in leather processing,detergent production industry and environmental protection.The whole genome of Thermoactinomyces vulgaris CDF was sequenced by three-generation sequencing technology,and the protease was predicted and analyzed.A total of 92 proteases were predicted,including 16 extracellular proteases,25transmembrane proteases,and 51 intracellular proteases.Thermoactinomyces vulgaris CDF is rich in protease resources,especially extracellular proteases have great potential for application.We used gelatin overlay assay to analyze the proteases in the extracellular medium of Thermoactinomyces vulgaris CDF and identified four proteases,three of them have been studied in previous work,including protease C2,TS-GSE and Als.Comparing the extracellular protease components of the log growth phase and the stationary phase of the CDF strain,it was found that one protease was present only in the logarithmic growth phase of the CDF strain.It is speculated that this protease plays an important physiological role in the growth of CDF strains.Mass spectrometric detection confirmed that it was the M6 family metalloproteinase and was named Tv M6.The DNA fragment encoding the Tv M6 was cloned and then introduced into Escherichia coli BL21(DE3)for expression.The Tv M6 precursor was obtained by Ni²⁺-NTA affinity chromatography and the Tv M6 precursor did not have proteolytic activity.The Tv M6 precursor can form an intermediate with protease activity by autoprocessing,but other proteases are required for further processing to form a mature enzyme.The mature form of Tv M6 was purified and characterized.The mature Tv M6displayed a temperature optimum of 65?,the p H optimum of 7.0-7.5 and retained approximately 80%of its original activity after incubation at 55C for 8 h.Calcium ions can increase the stability of mature Tv M6s while sodium chloride causes a decrease in the stability of mature Tv M6s.The mature Tv M6 can be further processed to from two polypeptide chains which have intermolecular interactions.These two polypeptide chains can form a structure which has protease activity.Studying the properties of Tv M6 not only developes new protease resources but also lays a foundation for studying the physiological functions of Tv M6 in Thermoactinomyces vulgaris CDF.