Enzymatic Properities Of Haloarchaeal Extracellular Protease Hlya And Its Preliminary Application In Fish Sauce Fermentation

Due to their characteristics of haloalkaliphilicity,good resistance to high temperature and organic solvents,extracellular proteases from haloarchaea have great application potentials in various industries.This study focused on the extracellular protease Hly A from a low-salt adapted haloarchaeon Halococcus salifodinae.The Hly A stability at different temperature,salinity and p H and its degradation products were investigated.The enzymatic properties of Hly A and its C-terminal extension（CTE）deletion mutant Hly A△CTE were compared.The effect of Hly A△CTE with desirable properties as an external enzyme on fish sauce fermentation was preliminarily studied.Hly A underwent autocleavage to produce a stable degradation product under high temperature or low salinity conditions.Hly A was obtained with high purity by heterologous expression in Escherichia coli,nickel affinity chromatography,in vitro refolding and gel filtration chromatography.Hly A displayed good stability at a relatively low temperature,high salinity or p H 6.0-10.0.It underwent autocleavage to produce a stable degradation product at a temperature above 40℃or salinity below 2 M Na Cl.C-terminal sequencing of the degradation product showed that it was the CTE deletion mutant of Hly A,namely,Hly A△CTE with only a catalytic domain.The purified recombinant Hly A△CTE precursor failed to refold into active enzyme.This result indicated that CTE played an important role in the autocatalytic maturation of Hly A precursor.The site-directed mutation of the autocleavage sites（P1 and P1’,Hly AE421A and Hly AE421A+T422A）did not pose a significant effect on the extracellular proteolytic activities of recombinant Haloferax volcanii strains.The refolded mutants also underwent autocleavage.Hly A△CTE possessed the properties of high stability,high activity and good tolerance to a wide range of conditions.The degradation product of Hly A,Hly A△CTE,was stable in a wide range of temperature（-20-60℃）,salinity（0.5-4 M Na Cl）and p H（6.0-10.0）.The optimum reaction conditions for Hly A were 40-45℃,p H 8.5,and those for Hly A△CTE were 60℃,p H7.0.Hly A and Hly A△CTE preferred low salinities（0.5-1.5 M Na Cl）.Hly A△CTE displayed better tolerance to temperature,and displayed higher activities under high temperature than Hly A.K⁺,Ca²⁺,Mg²⁺and Sr²⁺promoted the activities of Hly A and Hly A△CTE.Hly A and Hly A△CTE were able to hydrolyze different protein substrates,and showed good tolerance to DMSO and tween 20.The V_maxof Hly A△CTE was 2.1 times that of Hly A.Hly A△CTE could enhance proteolysis of fish protein and increase the free amino acid content in fish sauce.During the simulated fermentation process of fish sauce,the amino acid nitrogen content,polypeptide content and protease activity were all higher in the fish sauce with Hly A△CTE as an exogenous enzyme,than those with alkaline protease,with neutral protease,or without exogenous enzyme.The content of essential amino acids and umami amino acids was the highest in the fish sauce with Hly A△CTE.The total free amino acid content in the fish sauce with Hly A△CTE was 1.4 times that of the alkaline protease group,1.9 times that of the neutral protease group,and 3.0 times that of the control group without protease,respectively.In conclusion,Hly A underwent autocleavage at high temperature or low salinity conditions.The degradation product Hly A△CTE possessed desirable enzymatic properties.It could enhance hydrolysis of fish protein during fish sauce fermentation.This study would provide an important theoretical basis for the application of haloarchaeal extracellular proteases in high-salt fermented foods.