Purification, Characterization And Hydrolysis Specificity Of Protease From Deep-Sea Strain Exiguobacterium Sp. SWJS2

Controlling hydrolysis of protein by protease to produce functional proteins, peptides,and flavor condiments has become an important way to realize high-value utilization ofprotein resources. As the enzyme hydrolysis technology becoming mature and the rapiddevelopment of controllable protein enzymatic hydrolysis industry, there is an increasinglydemand for exploring proteases with novel characteristics. The deep-sea environment iscommonly subject to low temperature, faint sunshine, oligotrophic, high pressure and highsalt content. Marine microorganisms adapted to the condition often have obvious differencesfrom terrestrial counterparts in species, genetic composition, ecological function, metabolismsystem and defense system, which make them good sources of new bioactive substances withspecial structures and functions. This paper was aimed at isolating novel protease-producingbacteria from the deep-sea sediments, and studying on the enzymatic characteristics andhydrolysis specificity of the protease.Among the protease-producing bacterial strains isolated from deep-sea sediments,SWJS2was finally selected and identified as genus Exiguobacterium. Plackett-Burmandesign and orthogonal array design were applied to optimize the fermentation conditions andthe results were as followed: Glucose5g, yeast extract15g, glycerin2g and CaCl2·2H2O1gdissolved in1L artificial seawater; temperature25oC, original pH7, inoculum rate2%, seedage12h, medium volume25/250mL, shaking speed150rpm and fermentation time44h.The maximum protease production under the optimized parameters was702U/mL. It wasmuch higher than those of previously reported protease-producing Exiguobacterium genus.The crude protease was then purified by ultrafiltration, DEAE-Sepharose fast flowcolumn and Sephadex G-75column. The protease was finally purified to4.0-fold withspecificity activity of30654.1U/mg protein and a total yield of16.2%. The purified proteinshowed a single band with an apparent molecular weight of36kDa on SDS-PAGE. The Kmand Vmaxvalues for casein were7.9mg/mL and44.4μg/(mL·min). The enzyme was mostactive at40-45oC and pH7. It was heat-sensitive, losing all the activity after incubation attemperature higher than40oC for1h. Its thermostability was more close to cold-adaptedenzymes. The activity of the enzyme kept stable after being incubated in pH5-9buffer for24h. Cu2+, Ba2+, Cd2+, Hg2+and Al3+reduced the enzyme activity at5mM. Mg2+(0.5-2.5mM) and Mn2+(0.5-5mM) increased the protease activity by10%.1mM Ca2+couldincreased the protease activity by15%. The enzyme was totally inactivated at the existence of1or5mM EDTA but not affected by PMSF or DTNB, suggesting that it belonged to metal protease.The hydrolysis specificity of the newly developed protease from Exiguobacterium sp.SWJS2(EP) was evaluated based on the released free amino acid (FAA) during enzymolysisand through comparing with commercially available Papain and Alcalase2.4L. Resultsshowed that EP had great potential in producing hydrolysates with better nutrition and lessbitterness. The percentages of essential amino acids in the EP-treated Coilia mystus andsoybean protein were72%and70%, respectively. And corresponding hydrophobic aminoacids were74%and72%, respectively (i.e.<60%for Papain-or Alcalase2.4L-treatedsamples). The differences in FAA releasing rates between EP and the two commercialproteases suggested that EP could become a new commercial protease that offers differentreaction rates and extends protease application scope. Track changes in FAA profilesthroughout EP hydrolysis revealed the release of each amino acid exhibited its distinctregularity as the hydrolysis proceeded, which also varied with the substrate proteins. EPhydrolysis of Coilia mystus and soybean protein led to greater production rates of Phe, Leu,Val, Ile and Ala, suggesting that EP might have higher preference to these amino acids.