Characterization of a Recombinant Thermostable Arylsulfatase from Deep-Sea Bacterium Flammeovirga pacifica.
A novel sulfatase gene, ary423 (1,536 bp ORF), encoding a protein of 511 amino acids with a calculated molecular mass of 56 kDa, was identified from Flammeovirga pacifica, which was isolated from deep-sea sediments of west Pacific Ocean. Amino acid sequence analysis revealed that Ary423 possessed a conserved C-X-A-X-R motif, which was recognized as the sulfatase signature. Phylogenetic analysis suggested that Ary423 belonged to arylsulfatases. After heterologous expression in Escherichia coli cells, the recombinant Ary423 was purified with a Ni(+) affinity column, and was shown to be highly active at a broad range of temperatures from 30° to 70°C, with maximum activity at 40°C. Furthermore, recombinant Ary423 retained more than 70% and 40% of its maximum activity after 12 h of incubation at 50°C and 60°C, respectively, exhibiting good thermostability at high temperatures. The optimal pH for Ary423 was determined to be 8.0 and the activity of Ary423 could be slightly enhanced by Mg(2+). The recombinant enzyme could hydrolyze sulfate ester bonds in pnitrophenyl sulfate (NPS) and Asparagus crude polysaccharides with a specific activity of 64.8 U/mg and 25.4 U/mg, respectively. These favorable properties could make Ary423 attractive for application in the desulfating process of agar production.
XingHuaLing District Food and Drug Administration, Taiyuan 030000, P.R. China.
Chao
C
Gao
State Key Laboratory Breeding Base of Marine Genetic Resource; Third Institute of Oceanography, SOA, Xiamen 361005, P.R. China.
Min
M
Jin
State Key Laboratory Breeding Base of Marine Genetic Resource; Third Institute of Oceanography, SOA, Xiamen 361005, P.R. China.
Zhiwei
Z
Yi
State Key Laboratory Breeding Base of Marine Genetic Resource; Third Institute of Oceanography, SOA, Xiamen 361005, P.R. China.|Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen 361005, P.R. China.|South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510000, P.R. China.
Runying
R
Zeng
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001192:Arylsulfatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D041963:Bacteroidetes / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019015:Geologic Sediments / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009578:Nitrobenzenes / Q000378:metabolism; D010140:Pacific Ocean; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D020536:Enzyme Activators; D009578:Nitrobenzenes; D011134:Polysaccharides; D011994:Recombinant Proteins; C023379:4-nitrophenyl sulfate; D001192:Arylsulfatases; D008274:Magnesium
Flammeovirga Pacifica; arylsulfatase; characterization; deep sea; thermostability