Isolation Of An Arylsulfatase-producing Marine Bacterium And Characterization Of Its Arylsulfatase

Arylsulfatase hydrolyzes arylsulfate ester bonds to the corresponding phenolsand inorganic sulfate. The enzyme has the ability to degrade the sulfate group of agar,which can be applied to produce agrose replacing the current chemical methods. Inthis paper, a marine bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate and agar was isolated from the coastal area of QingDao. The strainis Gram-negative, aerobic and rod-shaped. Based on the phylogenetic and16S rDNAsequences analysis, the strain was classified named as Marinomonas sp. FW-1.After confirmed its ability of secreting arylsulfatase, the p-nitrophenyl was usedas the substrate to measure the quantitative of enzyme. In order to increase theenzyme activity, the single-factor experiments and response surface method wereadopted to optimize the culture conditions and medium composition. The optimalmedium compositions were agar of0.2%, yeast of0.2%, peptone of0.5%, NaCl of2.89%, MgCl2·6H20of0.412%, KCl of0.1%, CaCl2of0.02%, K2HPO4·3H20of0.013%, FeCl2·4H2O of0.002%, MnCl2of0.09%, Na4P2O7·10H2O of0.043%, andTW-80of1.87%. When cultivated in50mL medium of250mL shake flask, with theinoculum ratio of1%, the initial pH of7.0,35℃, and shaking speed of140rpm, thestrain Marinomonas sp. FW-1showed the highest activity of1.07U/mL at72h, whichwas4.28times higher than that before the optimization.Marinomonas sp. FW-1produced arylsulfatase steadily, and most intracellularly.The fermentation broth was collected by cultivated under the optimal cultureconditions and medium compositions. The arylsulfatase was partially purified bycryogenic centrifugal, ultrasonic cell disruption, ammonium sulfate precipation,dialysis desalination, ultrafiltration concentration, and native-polyacrylamide gelelectrophoresis. The molecular weight of arylsulfatase was determined to be60kDa.The optimal temperature and pH of the enzyme were45℃and11, respectively. Thearylsulfatase exhibited excellent thermostability and was perfact stable in low-temperature environment. Its activity kept95%after72h incubation under4℃, and kept90%after48h incubation under25℃. But when the temperatureexceed35℃,its activity decreased immediately after6h. The arylsulfatase also had a good stabilityunder pH7.0~11.0. With concentration of0.1mM/L, K+、 Ba2+, and Ca2+significantly increased the enzyme activity, while Hg2+inhibited it by59%.Furthermore, With concentration of0.1mM/L, Ba2+、Fe3+、Fe2+、Mg2+, and Ca2+also significantly increased the enzyme activity, while Hg2+inhibited it more by80%.The partially purified arylsulfatase was applied to degrade the sulfate of agar, and theresult indicated that, the rate of removal the sulfate group in Gelidium amansii andGracilaria agar were86.11%and89.61%, respectively. The results of this study laida good foundation for the further application of enzymaticaly production of agaroseby arylsulfatase.