Enhancement Of The Enzyme Activity Of Escherichia Coli RB3 Acetyl Esterase By A Strategy Of Tandem Repetitive Promoters

Acetyl esterase is an important members of carbohydrate esterase family(CE family) and it is also an important enzyme in xylanolytic enzymes system. Acetyl esterase plays an important role in degradation of biomass, papermaking and pharmaceutical industry. The yield of acetyl esterase cannot meet demand and researches for enhancement of acetyl esterase expression level are rare at present. As a result, finding an effective strategy to improve the yield in industrial production is necessary. In this study we tried to improve acetyl esterase yield of Escherichia coli applying strategy of tandem repetitive promoters. Firstly, yba C gene was amplified and sequenced from E. coli RB3, and a cloning vector p T-yba C was constructed. Secondly, we constructed an expression vector p K-yba C and obtained engineering bacteria of one copy tac promoter named ECON. Thirdly, we constructed engineering bacteria comtaining 2 copies(ECTW), 3 copies(ECTH) and 5 copies(ECFI) tac promoters. For researching the capacity of all strains, assays for acetyl esterase activity and transcriptional level of yba C gene were made. Finally acetyl esterase was purified from crude enzyme and characters of the enzyme were researched. We got conclusions as follows:(1) Referring to the sequences of yba C gene on NCBI primers for E. coli RB3 yba C were designed by Primer 5.0. Finally E. coli RB3 yba C gene was amplified via the optimization of PCR conditions. The result had been released on NCBI(Gen Bank accession No. KM489072).(2) Using E. coli JM110 as a host, ECON with one copy tac promoters inside was successfully constructed by the construction of p T-yba C and p K-yba C. Sequencing and enzyme cleavage was performed for verification.(3) By design and synthesis of tandem repetitive promoters we constructed multi-copy promoter plasmids with 2, 3 and 5 tac promoters inside. They were named p KTW-yba C, p KTH-yba C and p KFI-yba C. After they transformed E. coli JM110 we constructed ECTW, ECTH and ECFI successfully. Sequencing was also performed for verification and all strains were preserved at the condition of-70 °C. Growth curves were made for all constructed strains by assay of concentration of bacteria. Comparative analysis for the four strains were also made.(4) By drawing the standard curve of p-nitrophenol the assays of acetyl esterase activity were finished and the highest enzyme activity was calculated out for each strain. The result was that E. coli RB3 at condition of aerobic fermentation(0.367U/m L), ECON induced(48.65 U/m L), ECTW with no induction(28.32 U/m L), ECTH(3.04 U/m L) and ECFI(35.66 U/m L). ECTW showed the best capacity of producing acetyl esterase and ECFI had shortest fermentation period.(5) Total RNA was isolated from bacteria cells and synthesis of c DNA from m RNA was carried out. The m RNA level of yba C genes in strains was normalized by 16 S r RNA. The result showed that strains which had higher production level of acetyl esterase owned higher m RNA level. This meant the difference production level of acetyl esterase was attributed to different transcription level of yba C gene.(6) Anion-exchange chromatography was used to get purified acetyl esterase. Assay for characters of the enzyme from ECON and ECTW was carried out. Finally the data was analyzed and contrasted with different strains. The result showed AE from ECON and ECTW had similar characters. The optimal temperature was 51℃, optimal p H was 8. Zn2+, Ca2+and Ni2+ had auxo-action for acetyl esterase while the optimal concentration of metal ions was Ni2+(0.5 mmol/L).