Characterization Of Esterases From Bacillus Sp.And Their Performance In Phthalate Esters Degradation

Bacillus sp. HJ14 and Bacillus sp. SD01, Bacillus sp. SD02 were isolated from Yunnan and Shandong specific environment soil samples respectively. From the analysis results of complete genome sequencing and annotation we found that the genome sequence contains three sections of esterase gene Est Z1, Est Z14 and Est Z22. They were all successful cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant enzymes Est Z1, Est Z14 and Est Z22 were purified. We also characterized them and tried to determine their potential use for biodegradation of phthalate esters.(1) Recombinant Est Z1 hydrolyzed short-chain p NP esters(C2–C10) efficiently with the highest activity toward p-NPC4 according to initial velocity Kcat/Km values. No significant esterase activity was observed for the substrates with a chain length ≥10, indicating that Est Z1 is an esterase. Est Z1 showed highest activity at p H 9.0 and remained more than 60% activity of the original activity after incubated for 1 h in the buffer solution of p H 5.0–9.5. The optimal temperature for Est Z1 activity was 50 °C and was thermostable under the temperature of 37 °C and 50 °C, the half-life was 20 min when the temperature reached to 80 °C. Most of metal ions, chemical agents have little impact on it. Est Z1 could hydrolyzed diethyl phthalate(DEP) and diisobutyl phthalate(Di BP) to the corresponding monoalkyl phthalates.(2) Recombinant Est Z14 hydrolyzed short-chain p NP esters(C2–C6) efficiently with the highest activity toward p-NPC4 according to initial velocity Kcat/Km values. No significant esterase activity was observed for the substrates with a chain length ≥6, indicating that Est Z14 is an esterase. Est Z14 showed highest activity at p H 7.0 and remained more than 60% activity of the original activity after incubated for 1 h in the buffer solution of p H 6.0–11.0. The optimal temperature for Est Z14 activity was 45 °C and it was thermostable under the temperature of 37 °C, but its activity decline to less than 20% when the temperature was higher than 50 °C or less than 25 °C. Most of metal ions, chemical agents have little impact on it.(3) Recombinant Est Z22 hydrolyzed short-chain p NP esters(C2–C8) efficiently with the highest activity toward p-NPC4 according to initial velocity Kcat/Km values. No significant esterase activity was observed for the substrates with a chain length ≥8, indicating that Est Z22 is an esterase. Est Z22 showed highest activity at p H 9.0 and remained more than 60% activity of the original activity after incubated for 1 h in the buffer solution of p H 6.0–9.0. The optimal temperature for Est Z22 activity was 70 °C and was thermostable under the temperature of 37 °C, the half-life was 40 min and 15 min when the temperature reached to 60 °C and 80 °C respectively. Most of metal ions, chemical agents have little impact on it. Est Z22 had hydrolytic activity on DBP、DEP、Di BP and DCHP.In summary, this study got three esterases Est Z1, Est Z14 and Est Z22, Est Z1 and Est Z22 showed good thermostability and also could hydrolyzed DEP, Di BP, DBP, and DCHP. Thermophilic esterase has a great application potential in the food industry, biomedical and environmental management base on its dual nature of esterase and thermophilic enzyme.