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Cloning, Overexpression and Characterization of the Phytase Gene (PhyA)from Aspergillus Fumigatus Sp.

作　者: PHAN TRUNG HIEU
导　师: FAN LI QIANG
学　校: 华东理工大学
专　业: 生物化学
关键词: A.fumigatus phytase gene (PhyA gene) cloning expression Inclu-sion bodies Refolding
分类号: Q78
类　型: 硕士论文
年　份: 2012年
下　载: 9次
引　用: 0次
阅　读: 论文下载

内容摘要

Phytate, myo-inositol1,2,3,4,5,6-hexakisphosphate, is the primary source of inosi-tol and the major storage form phosphorus in agricultural feedstuffs, such as cereals, leg-umes, and oilseeds. Phytase, myo-inositol hexakisphosphate hydrolase (EC3.1.3.8for3-phytase and3.1.8.26for6-phytase), catalyzes the hydrolysis of phytate into myo-inositol and inorganic phosphates. Monogastric animals such as pigs and chickens are incapable of digesting phytate phosphorus due to the lack of, or low levels of, phytase activity in their digestive systems. Supplementation of phytase in these animals’feedstuffs enhances not only the nutritional quality of phytate-rich feed, but also the growth performance of the animals, thus decreasing phosphorus pollution in areas of intensive animal agriculture. The potential industrial applications of phytase increase the interest of isolation of new bacte-rial strains producing novel and efficient phytases is increasing.In this thesis, a phytase producing Aspergillus fumigatus sp (A. fumigatus sp) specie, was isolated and identified, the gene phyA encoding its thermostable phytase was cloned and heterologously expressed in E.coli, active recombinant phytase was purified from in-clusion bodies and then its thennostability and the pH dependence were assayed in detail.Firstly, isolation of A. fumigatus phytase producing was selected by size of clear zone on the plate with modified phytase screening medium.Secondly, total RNA was extracted from A. fumigatus grown on modified phytase screening medium, the cDNA libraries were generated by RevertAidTM First Strand cDNA Synthesis kit. The gene encoding phytase of A. fumigatus without signal peptide and intron, PhyA, which comprises1,329bp and encodes443amino-acid residues, was PCR amplified and sequenced. The nucleotide sequence of A. fumigatus phytase gene showed99%identities to that of A. fumigatus Af293phytase and67%homology to that of A. niger phytase. The deduced amino acid sequence of A. fumigatus sp. phytase showed65%and41%identities to those of A. terreus A91phytase and A. oryzae RIP40phytase, respectively. The phytase of A. fumigatus was predicted to be a novel member of the his-tidine-acid phosphatase family with its conserved motifs active site septa-peptide RHGXRXPT and catalytically active dipeptide HD in the amino-acid sequence.Thirdly, expression plasmid pET21a (+)-phyA was constructed by cutting PhyA of A. fumigatus without original leader peptide and intron from cloning vector pMD18T-phyA with Nde Ⅰ and EcoR Ⅰ and religated into the same restriction sites of prokaryotic ex- pression vector pET21a (+). The recombinant phytase was overexpressed in E. coli BL21(DE3) as an inclusion body after IPTG induction. However, numerous attempts, such as changing IPTG concentration, Ca2+concentration, or induction temperature, to improve the soluble expression of recombinant PhyA in E.coli were not successful. The molecular weight of expressed phytase was estimated as45kDa by SDS-polyacrylamide gel electro-phoresis.At last, inclusion body renaturation conditions and partial characterics of recombi-nant phytase were studied. Ca2+ion was essential for obtaining active enzyme. Recombi-nant A. fumigatus phytase with specific activity of625UmL-1was obtained by refolding at pH5.5acetate buffer including1mM Ca2+. Purified phytase exhibited optimal activity at60℃. The enzyme retained70%of its original activity after20min incubation at80℃. Optimum pH was5.5-6.0. Recombinant phytase was more stable at acid conditions than neutral and alkaline conditions. It remained fairly stable over pH range of5.5to6.5.The broad pH optima and high thermostability of the phytase makes it a promising candidate for feed-pelleting application.

全文目录

TABLE OF CONTENTS  5-9
LIST OF FIGURES  9-11
LIST OF TABLES  11-12
ABSTRACT  12-14
CHAPTER 1. INTRODUCTION  14-33
  1.1. Phytate and phytases  15-19
    1.1.1. Phytate  15-17
    1.1.2. Phytase  17-19
  1.2. Sources of Phytase  19-24
    1.2.1. Microbes  19-21
      1.2.1.1. Fungal source  19-20
      1.2.1.2. Bacterial source  20-21
      1.2.1.3. Yeast source  21
    1.2.2. Plant source  21-22
    1.2.3. Animal sources  22-24
  1.3. Recombinant phytase expression  24-25
  1.4. Structure  25-30
    1.4.1. Histidine acid phosphatases (HAPs)  25-27
    1.4.2. β-propeller phytases (βPP)  27
    1.4.3. Cysteine phosphatase (CPs)  27-28
    1.4.4. Purple acid phosphatases (PAPs)  28-30
  1.5. Application of phytase  30-32
    1.5.1. Production of phytase in transgenic plants  30
    1.5.2. Feed application  30-31
    1.5.3. Food application  31-32
    1.5.4. Pulp and paper industry  32
  1.6. Scope of the thesis  32-33
CHAPTER 2. SCREENING, CLONING AND SEQUENCING OF PHYTASE FROM ASPERGILLUS FUMIGATUS  33-47
  2.1 Materials  33-34
    2.1.1 Stains and vectors  33
    2.1.2 Apparatus  33
    2.1.3 Reagents  33-34
  2.2 Methods  34-38
    2.2.1 Microorganism and growth media  34
    2.2.2 RNA isolation from Aspergillus fumigatus  34-35
    2.2.3 cDNA synthesis by reverse transcriptase polymerase chain reaction (RT-PCR)  35
    2.2.4 Amplification of phytase gene  35-36
    2.2.5 Construction of cloning vector pMD18T-phyA  36
    2.2.6 Preparation of competent cells  36-37
    2.2.7 Transformation  37
    2.2.8 Preparation of plasmids  37-38
    2.2.9 Identification of positive recombinant clones  38
    2.2.10 DNA sequence and computer analysis  38
  2.3 Result and discussion  38-47
    2.3.1 Screening for phytase-producing from A. fumigatus  38-39
    2.3.2 Total RNA Isolation and Reverse Transcription  39-40
    2.3.3 PCR amplification of phytase gene from A. fumigatus  40-41
    2.3.4 Construction of cloning plasmid pMD18T-PhyA  41-42
    2.3.5 Sequence Analysis  42-47
CHAPTER 3. EXPRESSION AND OPTIMIZATION OF PHYTASE GENE (PhyA) IN ESCHERICHIA COLI  47-54
  3.1 Material  47
    3.1.1 Bacterial strain and plasmid  47
    3.1.2 Reagents  47
  3.2 Methods  47-50
    3.2.1 Construction of expression vector pET21-phy A  47-48
    3.2.2 Transformation of competent Escherichia coli  48
    3.2.3 Expression of pET21-phyA gene in E. coli  48
    3.2.4 Phytase activity assay  48-49
    3.2.5 Total protein assay  49-50
    3.2.6 Optimization of phytase expression  50
  3.3 Results and discussion  50-54
    3.3.1 Construction of prokaryotic expression vector pET21-phy A  50-51
    3.3.2 Effect of incubation temperature  51
    3.3.3 Effect of IPTG concentration  51-52
    3.3.4 Effects of Ca2+ ion concentration  52-54
CHAPTER 4. CHARACTERIZATION OF PHYTASE (PhyA) AS INCLUSION BODIES IN E.COLI  54-61
  4.1 Materials  54
    4.1.1 Bacterial strains  54
    4.1.2 Reagents  54
  4.2 Methods  54-56
    4.2.1 Expression of recombinant phytase  54
    4.2.2 Isolation of inclusion bodies  54-55
    4.2.3 Solubilization and refolding of PhyA proteins  55
    4.2.4 Dialysis  55-56
    4.2.5 Concentration with Poly Ethylene Glycol (PEG) 20,000  56
    4.2.6 Effects of temperature and pH on phytase activity and stability  56
      4.2.6.1 Optimum temperature  56
      4.2.6.2 Optimum pH  56
  4.3 Result and Discussion  56-61
    4.3.1 Expression of recombinant pET21a-PhyA in E.coli  56-58
    4.3.2 Effects of temperature and pH on phytase activity  58-61
      4.3.2.1 Optimum temperature and thermostability  58-59
      4.3.2.2 Optimum pH and pH stability  59-61
CHAPTER 5. CONCLUSION  61-63
REFERENCES  63-70
APPENDICES  70-78
  APPENDIX Ⅰ. Czapek-Dox agar medium for screening phytase  70
  APPENDIX Ⅱ. cDNA synthesis by reverse transcriptase polymerase chain reaction  70-71
  APPENDIX Ⅲ. Reaction mix for PhyA amplification  71
  APPENDIX Ⅳ. Agarose gel electrophoresis  71-72
  APPENDIX Ⅴ. Ligation recipes  72-73
  APPENDIX Ⅵ. Ligation of PhyA insert and pET21a  73
  APPENDIX Ⅶ. Reagents for plasmid isolation  73-74
  APPENDIX Ⅷ. Sodium Dodecyl Polyacrylamide Gel Electrophoresis(SDS-PAGE)  74-76
  APPENDIX Ⅸ. Determination of Phytase Activity-Molybdate-Blue Method(Fujian Fuda Biotech Co., Ltd.)  76-78
ACKNOWLEDGEMENTS  78

Cloning, Overexpression and Characterization of the Phytase Gene (PhyA)from Aspergillus Fumigatus Sp.

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