Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197.
A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The protein sequence of Est-gela shows similarity with β-lactamases and esterases (⩽ 43%). Est-gela contains the Ser-X-X-Lys conserved sequence (Ser58-Met59-Thr60-Lys61) and belongs to family VIII of esterases. We overexpressed Est-gela in Escherichia coli XL1-blue and purified this protein using a His tag. Est-gela showed a strong enzymatic activity toward p-nitrophenyl esters with short acyl chains (⩽ C4) and the strongest activity toward p-nitrophenyl butyrate. Est-gela showed an enhanced enzymatic activity at 65-75 °C and retained more than 90% of the activity after incubation at 65 °C for 180 min. These results indicated that Est-gela was thermostable. In addition, Est-gela showed the maximal activity at pH 10. We also evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. Finally, Est-gela hydrolyzed (R,S)-ketoprofen ethyl ester (Kcat/Km = 5.0 ± 0.2 s(-1) mM(-1), mean ± standard error) with enantioselectivity toward (S)-ketoprofen ethyl ester rather than (R)-ketoprofen ethyl ester.
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea.
Jinyeong
J
Kim
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea.
Lili
L
Deng
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea.
Eunsoo
E
Hong
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea. Electronic address: ywryu@ajou.ac.kr.
Yeonwoo
Y
Ryu
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D004950:Esterases
Bacillus gelatini; Characterization; Esterase; Screening; Thermostability