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Molecular Cloning, Expression and Characterization of a Thermophilic Carboxylesterase from Dictyoglomus Turgidum

Author: ZhengCuiHong
Tutor: FengYan
School: Shanghai Jiaotong University
Course: Biophysical
Keywords: Dictyoglomus turgidum Carboxylesterase Cloning Expression Characterization
CLC: Q55
Type: Master's thesis
Year: 2012
Downloads: 65
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Abstract

Carboxylesterases （EC 3.1.1.1） are enzymes that catalyze the hydrolysis of carboxylic ester into carboxylic acids and alcohols, which are nonspecific esterases. The structure of carboxylesterases belongs toα/βhydrolase fold family and bears a catalytic triad formed by Ser-Asp-His in the active site. Carboxylesterases from microorganisms can effectively catalyze the hydrolysis of esters and acylamides, the synthesis of esters and interesterification with good regioselectivity and enantioselectivity, thus have a wide application prospects in food, biological detergent, medicine, energy development, environment protection and related industries.Thermophilic carboxylesterases from thermophiles have good thermostability and high resistance to organic solvents and chemical denaturant, showing unique advantages compared with the conventional carboxylesterases. Consequently, they become one hotspot in the biotechnology research field in recent years.Through a series of comparision and analysis of the genome database, we find a putative carboxylesterase gene （Dtur₀242） in thermophilic bacteria Dictyoglomus turgidum DSM 6724 genome encoding 412 amino acids. It has been predicted that Dtur₀242 gene may encode a carboxylesterase or phospholipase, whose theoretical mass and isoelectric point are 46874.21 Da and 5.82 respectively. Through comparison with homologous protein sequence, we find that the target protein contains a catalytic triad formed by Ser-Asp-His triad and a conserved pentapeptide Gly-Xaa-Ser-Xaa-Gly. Compared with lipolytic enzymes representing eight different families, the target protein has low homology with them. But it clustered with the lipolytic enzymes from familyⅧ. So the target protein has good research significance because of its independent evolution relationship.We successfully cloned the Dtur₀242 gene and constructed the engineering bacteria Escherichia coli BL21 （DE3） CodonPlus-RIL containing the recombined plasmid pET-28a + Dtur₀242. Through breaking the bacteria, heating denaturation and Ni-NTA purification, we got the purified enzyme, and the recovery rate reached 65.0%. Then, we named it DtEstA. It has been found that it has high carboxylesterase activity with little phospholipase activity. Its optimum temperature is 80℃and optimum pH is 8.0. Also it can hydrolyze both p-nitrophenyl esters and triglyceride of different carbon-chains, and the optimal substrate is p-nitrophenyl caprylate whose kcat is 352.61s-1 and Km is 333.6μM. Meanwhile it shows perfect stability under high temperature. When the concentration of DtEstA is 0.66mg/mL, the residual activity retains more than 85% after incubating at 65℃for 120h, and the t1/2 at 75℃is about 95 hours.Besides, the influence of metal ions, organic solvents and detergent on DtEstA activity was investgated. The results show that Fe²⁺, K⁺, Ca²⁺, Na⁺ and Mg²⁺ have activation on enzyme activity. When the concentration of Fe²⁺ is 0.2mM, the activation can reach 46.1%. Ni²⁺ and Zn²⁺ can inhibit the activity. When the concentration of Ni²⁺ is 5mM, the inhibition is 53.3%. When the concentration of Zn²⁺ is 5mM, the inhibition is 84.9%. Moreover, the recombinant DtEstA shows excellent abilities in resistance organic solvents and detergent. When the methanol concentration is as high as 20%, the residual activity is still 67.4%. And when the acetone concentration is 5%, 88% activity is retained. DtEstA retains 30% activity in the environment of 5% SDS. All merits make DtEstA can play important role in organic medium, food, pharmaceuticals and related fields. In conclusion, DtEstA is successfully cloned, expressed and characterized. Our research shows that DtEstA is a novel thermophilic carboxylesterase of high esterase activity, good thermostability and resistance against metal ions, organic reagents and detergent. Thus, DtEstA will have a bright prospect in the biochemical industry.

Molecular Cloning, Expression and Characterization of a Thermophilic Carboxylesterase from Dictyoglomus Turgidum

Abstract

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