Characterization of novel cold active and salt tolerant esterases with improved catalytic properties through immobilization

摘要

Esterase (EC 3.1.1.1) is a valuable industrial biocatalyst which hydrolyzes esters into alcohol and acid, or catalyzes ester formation through reverse reaction.Esterases show high enantion, regio-and stereo-selectivity towards its substrates.So this biocatalyst is studied widely and applied in different types of industrial applications such as in food,detergent, pharmaceuticals and chemical industries.Cold active esterases exhibit high catalytic efficiency at low temperature and could be energy saving and helpful for the synthesis of thermo labile compounds. Immobilization could increase the range of application of esterase through making it more stable to extreme conditions and benefits by reusability and fine chemical synthesis.Two novel esterase coding genes, EstLiu and EstH were cloned from the marine bacterium Zunongwangia profunda, overexpressed in E.coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography.The mature esterase EstLiu and EstH sequence encodes a protein of 273 and 547 amino acids residues, with a predicted molecular weight of 30 and 61.2 KDa.EstLiu contains the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243 and the conserved motif for the EstH is Gly 219-X-Ser 221-X-Gly 223 and the catalytic triads are Ser 221-Glu 337-His 456.Phylogenetic analysis showed EstLiu had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family and EstH belongs to family Ⅶ.Both purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). Though the optimal temperature 30℃ were same for both enzymes and quickened inactivation above 60℃, EstLiu showed remarkable activity (75％) at 0℃ and the optimal activity at pH 8.0 and EstH showed～50％ of original activity at 0℃ and pH 8.5.Both of them were stable in high salt conditions (0-4.5 M NaCl) with good performance in the presence of organic solvents and detergents.To improve the characteristics and explore the possibilities for application of EstH, a new immobilization matrix, Fe3O4～cellulose nano-composite, was prepared where the magnetic nanoparticles of Fe3O4 were synthesized by co-precipitation of Fe2+ and Fe3+ ions in ammonia solution and Fe3O4～cellulose nano-eomposite was prepared by the solgel method and was characterized by Fourier Transform Infrared Spectroscopy (FTIR)and Scanning Electron Microscope (SEM). Compare with free form, interestingly the optimal temperature of immobilized EstH elevated to 35℃. It showed better temperature stability (48.5％ compared to 22.40％ at 50℃ after 30 min), prolonged half-life (32h compared to 18h), storage stability (～71％ activity compared to～40％ activity after 50 days of storage), pH tolerance (～73％ activity at pH 4 and 10), and more importantly, reusability (～50％ activity after 8 repetitive cycles of usage).Enzyme kinetics showed an increase in the Vmax (from 35.76 to 51.14μM/min) and Kcat (from 365 S-1 to 520 S-1) after immobilization onto nano-composite. The superior catalytic properties of EstLiu and immobilized EstH suggest their great potential in bioteehnology and industrial processes.

目录

声明

Table of Contents

Abstract

Abbreviations

1 Introduction

1．1 Enzyme

1．2 Esterase

1．2．1 Structure of esterases

1．2．2 Catalytic mechanism of esterase／lipase

1．2．3 Interfacial activation of esterases／lipases

1．2．4 Classification of esterases／lipases

1．2．5 Applications of esterases／lipases

1．2．6 Detection of esterase activity

1．2．7 Sources of esterases

1．2．8 Psychrophiles

1．2．9 Enzyme Immobilization

1．2．10 Matrix for immobilization

1．2．11 Applications of immobilization

1．2．12 Purpose and significance of this research

2 Materials and methods

2．1 Materials

2．1．1 Strains and plasmids

2．1．2 Reagents

2．1．3 Medium

2．1．4 Solutions

2．1．5 Equipment

2．2 Cloning of esterase gene

2．2．1 Genomic DNA extraction of Zunongwangia Profunda

2．2．2 Esterase gene was amplified by PCR

2．2．3 PCR products purification and digestion

2．2．4 PGEX-6P-1 vector preparation

2．2．5 Ligation reaction

2．2．6 Preparation of E．coli competent cell

2．2．7 Electroporation

2．2．8 Detection of positive clones

2．3 Expression and purification of esterases

2．3．1 Expression and purification of EstLiu and EstH

2．3．2 Protein analysis by SDS-PAGE

2．3．3 Protein concentration determination

2．4 Enzymatic Properties Determination

2．4．1 Standard curve drawing

2．4．2 Enzymatic activity measurement

2．4．3 Determination of substrate specificity

2．4．4 Determination of optimum temperature

2．4．5 Determination of temperature stability

2．4．6 Optimal pH measurement

2．4．7 Determination of pH stability

2．4．8 Effect of metal ions and other chemicals on the activity of EstLiu

2．4．9 Effect of different organic solvents on the esterase activity

2．4．10 Effect of different detergents on esterase activity

2．4．11 Effect of high concentrations of NaCl solution in esterase activity and stability

2．5 Immobilization

2．5．1 Synthesis of Fe3O4～cellulose nano-composite

2．5．2 Characterization of Fe3O4～cellulose nano-composite

2．5．3 Immobilization of purified esterase onto Fe3O4～cellulose nano-composite

2．5．4 Determination of reusability and storage stability of immobilized EstH

2．6 Sequence analysis

2．7 Kinetic parameters

2．8 Structural modeling

3 Results

3．1 Genomic DNA Extraction of Zunongwangia Profunda

3．2 PCR amplification of esterase genes

3．2．1 PCR amplification of EstLiu

3．2．2 PCR amplification of EstH

3．3 Recombinant plasmid Construction

3．3．1 Recombinant plasmid of pGEX-6p-1-EstLiu Construction

3．3．2 Recombinant plasmid of pGEX-6p-1-EstH Construction

3．4 Sequence analysis of the esterases

3．4．1 Sequence analysis of EstLiu

3．4．2 Sequence analysis of EstH

3．5 Expression and purification of proteins

3．5．1 Expression and purification of EstLiu

3．5．2 Expression and purification of EstH

3．6 Synthesis and characterization of FesO4～cellulose nano-composite

3．7 Immobilization of purified esterase onto Fe3O4～cellulose nano-composite

3．8 Characterization of EstLiu and EstH

3．8．1 Product standard curve preparation

3．8．2 Determination of substrate specificity

3．8．3 Effect of temperature on the enzyme activity and stability

3．8．4 Effect of pH on the activity and stability

3．8．5 Effect of metal ions 011 the activity of esterase

3．8．6 Effect of organic solvents on the esterase activity

3．8．7 Effect of detergents and other chemicals on the esterase activity

3．8．8 Effect of NaCl on the activity and stability of esterase

3．8．9 Reusability assay of immobilized EstH

3．8．10 Storage ability determination of immobilized EstH

3．8．11 Kinetic parameters of esterase

3．8．12 Structural modeling of esterases

4 Discussion

5 Conclusion

References

Publications

Awards

ACKNOWLEDGEMENTS