[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

摘要

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.

材料

Product Number

品牌

产品描述

E7023

Sigma-Aldrich

乙醇，Pure 200纯度, Molecular Biology

459844

Sigma-Aldrich

纯乙醇, 200 proof, ACS reagent, ≥99.5%

459828

Sigma-Aldrich

纯乙醇, 200 proof

493546

Sigma-Aldrich

纯乙醇, 200 proof, meets USP testing specifications

459836

Sigma-Aldrich

纯乙醇, 200 proof, anhydrous, ≥99.5%

E7148

Sigma-Aldrich

乙醇，Pure 190纯度, for molecular biology

493511

Sigma-Aldrich

纯乙醇, 190 proof, ACS spectrophotometric grade, 95.0%

51976

Sigma-Aldrich

酒精, BioUltra, Molecular Biology, ≥99.8%, (absolute alcohol, without additive, A15 o¹)

270741

Sigma-Aldrich

酒精试剂, denatured, suitable for HPLC

362808

Sigma-Aldrich

酒精试剂, reagent grade

PHR1070

Supelco

无水乙醇, Pharmaceutical Secondary Standard; Certified Reference Material

493538

Sigma-Aldrich

纯乙醇, 190 proof, meets USP testing specifications

187380

Sigma-Aldrich

乙醇，变性, reagent grade, denatured

02857

Sigma-Aldrich

酒精, purum, fine spirit, denaturated with 4.8% methanol, F25 METHYL¹, ~96% (based on denaturant-free substance)

02483

Supelco

酒精, standard for GC

E-029

Supelco

乙醇-50标准液 CRM, 50 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-033

Supelco

乙醇-300标准液 CRM, 300 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

277649

Sigma-Aldrich

酒精试剂, anhydrous, ≤0.003% water

E-040

Supelco

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

E-030

Supelco

乙醇-80 标准液 CRM, 80 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-031

Supelco

乙醇-100（10支/盒)标准液 CRM, 100 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

32221

Sigma-Aldrich

酒精, puriss. p.a., absolute, ≥99.8% (GC)

676829

Sigma-Aldrich

酒精试剂, anhydrous, ≤0.005% water

48075

Supelco

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

29221

Sigma-Aldrich

酒精, tested according to Ph. Eur.

E2385

Supelco

10% (v/v) 乙醇标准品, 10 % (v/v) in H₂O, analytical standard

E-053

Supelco

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

E-041

Supelco

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

E-032

Supelco

乙醇-200 (10支/盒)标准液 CRM, 200 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®

E-036

Supelco

乙醇-400（10支/盒)标准液 CRM, 400 mg/dL in H₂O, ampule of 10 × 1.2 mL, certified reference material, Cerilliant^®