[Cloning, expression and characterization of alcohol dehydrogenase and aldehyde dehydrogenase from Bacillus pseudofirmus OF4].

[Cloning, expression and characterization of alcohol dehydrogenase and aldehyde dehydrogenase from Bacillus pseudofirmus OF4].

Wei sheng wu xue bao = Acta microbiologica Sinica (2013-07-19)

Jiansong Ju, Ning Ma, Ranran Zhao, Jingwei Liu, Shujing Xu, Baohua Zhao

ABSTRACT

To clone and characterize the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from Bacillus pseudofirmus OF4. Genes of adh and aldh were cloned by PCR; expression vectors pET-Ahd and pET-Aldh were constructed and expressed in Escherichia coli BL21 (DE3). After Ni-NTA column chromatography purification, the protein was characterized. The optimal temperature and pH of ALDH was 35 degrees C and 8.0, the specific activities of ALDH was 979.6 U/mg protein, the thermostability at 25 degrees C and 35 degrees C was better than at 45 degrees C. Although the expression level of ADH was too low to purify, but it was found that ADH had high catalytic activities by experiments of co-expression and ethanol tolerance. Adh and aldh from B. pseudofirmus OF4 were cloned successfully. Co-expression of double genes could greatly increase the host strain on ethanol tolerance.

MATERIALS

Product Number

Brand

Product Description

270741

Sigma-Aldrich

Reagent Alcohol, suitable for HPLC

PHR1070

Supelco

Dehydrated Alcohol, Pharmaceutical Secondary Standard; Certified Reference Material

E-056

Supelco

Ethanol-20 (10 ampules/kit), 20 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-053

Supelco

Ethanol-500, 500 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-035

Supelco

Ethanol-25, 25 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-036

Supelco

Ethanol-400 (10 ampules/kit), 400 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

277649

Sigma-Aldrich

Reagent Alcohol, anhydrous, ≤0.003% water

362808

Sigma-Aldrich

Reagent Alcohol, reagent grade

48075

Supelco

Ethanol solution, certified reference material, 2000 μg/mL in methanol

676829

Sigma-Aldrich

Reagent Alcohol, anhydrous, ≤0.005% water

2858

Sigma-Aldrich

Ethanol Fixative 80% v/v, suitable for fixing solution (blood films)

E-034

Supelco

Ethanol Calibration Kit, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

51976

Sigma-Aldrich

Ethanol, BioUltra, for molecular biology, ≥99.8%, (absolute alcohol, without additive, A15 o¹)

02483

Supelco

Ethanol, standard for GC

187380

Sigma-Aldrich

Ethyl alcohol, denatured, reagent grade

02891

Sigma-Aldrich

Ethanol, purum, secunda spirit, denaturated with 2% 2-butanone, S15, ~96% (based on denaturant-free substance)

02851

Sigma-Aldrich

Ethanol, for residue analysis

459836

Sigma-Aldrich

Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%

493511

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%

493538

Sigma-Aldrich

Ethyl alcohol, Pure, 190 proof, meets USP testing specifications

E2385

Supelco

Ethanol standards 10% (v/v), 10 % (v/v) in H₂O, analytical standard

E-041

Supelco

Ethanol-150, 150 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-045

Supelco

Ethanol-40, 40 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-040

Supelco

Ethanol-10, 10 mg/dL in H₂O, pack of 10 × 1.2 mL ampules, certified reference material, Cerilliant^®

29221

Sigma-Aldrich

Ethanol, tested according to Ph. Eur.

E-031

Supelco

Ethanol-100 (10 ampules/kit), 100 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-032

Supelco

Ethanol-200 (10 ampules/kit), 200 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

02882

Sigma-Aldrich

Ethanol, purum, absolute ethanol, denaturated with 2% 2-butanone, A15 MEK¹, ≥99.8% (based on denaturant-free substance)

02870

Sigma-Aldrich

Ethanol, ACS reagent, prima fine spirit, without additive, F15 o¹

02890

Sigma-Aldrich

Ethanol, purum, fine spirit, denaturated with 2% 2-butanone, F25 MEK¹, ~96% (based on denaturant-free substance)