TY - JOUR

T1 - Cloning, expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII

AU - Samoylova, Yuliya V.

AU - Sorokina, Ksenia N.

AU - Romanenko, Margarita V.

AU - Parmon, Valentin N.

PY - 2018/3/1

Y1 - 2018/3/1

N2 - A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

AB - A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

KW - Bacteria

KW - Esterase

KW - Solvent tolerance

KW - Thermostability

KW - Ureibacillus thermosphaericus

KW - CLASSIFICATION

KW - BACTERIAL RECOMBINANT LIPASE

KW - ALKALINE

KW - THERMOSTABLE ESTERASE

KW - 1ST MEMBER

KW - ENZYMES

KW - PSEUDOMONAS-FLUORESCENS CARBOXYLESTERASE

KW - PURIFICATION

KW - MOLECULAR-CLONING

KW - GENE CLONING

UR - http://www.scopus.com/inward/record.url?scp=85041691693&partnerID=8YFLogxK

U2 - 10.1007/s00792-018-0996-9

DO - 10.1007/s00792-018-0996-9

M3 - Article

C2 - 29330648

AN - SCOPUS:85041691693

VL - 22

SP - 271

EP - 285

JO - Extremophiles

JF - Extremophiles

SN - 1431-0651

IS - 2

ER -