Cloning, sequence analysis and
characterization of a novel i-glucosidase- like activity, MUGA from Pichia etchellsii
By
Pranita Roy
Department of Biochemical Engineering and Biotechnology
Submitted
in fulfillment of the requirements of the degree of
Doctor of Philosophy
to the
Indian Institute of Technology, Delhi
October 2005
Dedicated to my 6efoved parents
CE VI TI CA TE
This is to certify that the thesis entitled "Cloning, Sequence analysis and characterization of a novel jI-glucosidase MUGA from Pichia etchellsii', being submitted by Ms. Pranita Roy to the Indian Institute of Technology, Delhi, foi the award of the degree of "Doctor of Philosophy", is a record of the bonafide research carried out by her, which has been prepared under my supervision in conformity with rules and regulations of the "Indian Institute of Technology, Delhi". The research reports and results presented in the thesis have not been submitted for any degree or diploma in any other University or Institute.
Prof. Saroj Mishra
AC7C7■TOWLEDgEatovir
I take this pleasure to express deep sense of gratitude and indebtness towards my ph.D supervisor Prof Saroj Mishra for her expert guidance and encouragement throughout the course of this project. I would like to thank her for her critical and inspiring suggestions which helped me decide the right approach for every investigation and develop my research skills. I shall never fail to remember her being so supportive, for even lending her experimental hands whenever required. I will always be thankful to her for her optimism and patience which helped me develop more confidence.
A special acknowledgement is due to Dr. T. K. Choudhuri for his generous advice and suggestions which was of great benefit. I am also thankful to my SRC members Prof S. Chand and Dr. J. K. Deb for their invaluable feedback.
I sincerely thanks all the faculty and staff members of DBEB for their constant help and support. Special thanks are due to Mr. V. K. Ghosh who contributed his help in numerous ways. I shall never fail to remember his technical help and for making things available to me. I sincerely thanks Mr. S. Patra for his skilful help in preparation of this thesis. I also extend my thanks to Mr. M. Anand, Mr. Kishan and Mr. Ramgopal for their timely help by providing me with all equipments and chemicals.
Special thanks are due to my seniors Mrs. Yukti, Mrs. Shivani and Mrs. Anu for their help and suggestions at initial stages. I am also thankful to my friends Salony and Rumpa for their timely help and unconditional support and all my colleagues, Rupali, Mili, Anjali, Parul, Anand, Ranjita, and all others for their pleasant company and support. Special thanks are due to Snehasis for his generous help and Bhawna for always being there cheerfully and willingly lending a hand.
I owe deep indebtness towards Richa for her invaluable contribution and unconditional support. I shall never fail to remember her lending me a hand in some of the crucial experiments. My warmest thanks are due to her.
I express my heartfelt gratitude towards my mummy and papa for their constant love, affection and painstaking care which kept me going. I will always be grateful to them for believing in me and making me the person I am today. My warmest thanks are due to my sisters and brothers for their love and patiently putting up in my absence. I would like to extend my thanks to my parents in law for their moral support, recurring encouragement and being there for me through thick and thin.
Last but not the least, I express my heartfelt thanks to my husband Sunil for his patience, concern and constant emotional support when most needed. Without his care and undemanding attitude this thesis would have been just a dream. My warmest thanks are due to my daughter Prakriti for boosting up my sagging enthusiasm by her refreshing smile.
( Pranita Roy)
iii
ABSTRACT
0-glucosidases (3-D-glucoside glucohydrolase, EC 3.2.1.21) comprise a heterogenous group of enzymes that are able to cleave the /3-glucosidic linkages of di-and/or oligosaccharides and other glucose conjugates. These enzymes are widely distributed in the living world and play pivotal roles in many biological processes. To study the biological role and useful biotechnological applications of these enzymes, we have reported on a number of p-glucosidases isolated from the thermo-tolerant yeast Pichia etchellsii. This organism grows optimally at 40-45°C and produces multiple 0- glucosidases. Two enzymes namely, Bgl I and Bgl II were identified by way of expression in Escherichia coli. Two /3-glucosidases have been purified from the cell wall of the yeast, namely BGLI and BGLII and detailed analysis indicated these to be different from each other and from the other E. coli expressed enzyme. Our continued search in this yeast system has lead to identification of a novel hydrolytic activity. It resembled the reported 3-glucosidases in terms of its ability to hydrolyse MUG (methyl umbelliferyl 13- D-glucoside) but did not hydrolyze pNPG (p-nitrophenyl (3-D-glucoside), which is a commonly used substrate for assay of these enzymes. The present work was undertaken to determine and analyze the nucleotide sequence encoding this activity, characterize the purified protein (biochemically and structurally), and evaluate its function in the yeast.
Genomic DNA fragment encoding a noveli3-glucosidase-like activity of the yeast P. etchellsii was cloned and expressed in E. coli. Two MUG hydrolyzing clones were isolated, namely, pMG8: DH5a and pMG16: DH5a containing the same gene on different insert lengths. The sequencing of the 6.35 kbp yeast insert in pMG8 plasmid showed multiple ATG's with a single termination codon (TAG). An open reading frame
iv
(ORF) of 1515 by termed mugA was confirmed by sub-cloning the pMG8 plasmid which codes for a protein of predicted molecular mass of 54.1 kDa. The sequence homology search of the ORF did not show homology with any of the reported 0-glucosidases, however, a very significant identity was seen with several Ser (S)-Asp (D) rich cell surface proteins. The secondary structure prediction program 3D-PSSM indicated the protein to be composed of largely helical and coiled structures, quite characteristic of cell-surface associated proteins which was confirmed by circular dichroism spectroscopy.
The MUGA protein was found to be localized in the intracellular space of re-E. co/i. It was released by sonication and used as starting material for purification. The protein was purified to homogeneity by a combination of DEAE-Sepharose, hydroxyapatite and Sephadex G-25 column chromatography to 53 fold purity. The biochemical properties of purified MUGA were investigated in detail. The molecular mass of the protein determined from SDS-PAGE gel was around 50.1± 5.5 kDa while the mass was estimated to be 52.1 kDa by MALDI-TOF. The protein was optimally active at 45°C and in the pH range of 6-11. The protein displayed high hydrolytic activity on MUG but relatively very little hydrolysis of pNPG and gentiobiose, characteristic substrates for (3- glucosidases. Kinetic measurements indicated that the best substrate was MUG, with highest value of specificity constant ( kat/Km) of 0.083 x 10-3 gmol-lmin-1 . However, the protein displayed higher affinity towards pNPG. The protein did not show any stimulation or inhibition in activity in the presence of divalent ions, denaturants and group specific reagents. However, slight increase in hydrolytic activity was obtained in the presence of methanol.
v
The binding experiments performed between P. etchellsii cells and the purified E.
colt expressed MUGA indicated binding with the cell surface which was monitored by fluorescence microscopy. In competition experiments with the SD dipeptide, less protein was shown to bind to the cell surface, in a concentration dependent manner indicating the binding of MUGA protein to the cell-surface. The expression of the mugA gene in BL21 (DE3) was significantly improved by its positioning under control of the T7 promoter in the pET29a expression vector.
COICIE.WTS
Page No.
CERTIFICATE i
ACKNOWLEDGEMENT ii
ABSTRACT iv
CONTENTS vii
LIST OF FIGURES xii
LIST OF TABLES xv
LIST OF ABBREVIATIONS xvi
1. INTRODUCTION AND OBJECTIVES 1
1.1 Introduction 1.2 Objectives
2. REVIEW OF LITERATURE 10
2.1 Occurrence, function and properties of 13-glucosidases 2.1.1 Bacterial /3-glucosidases
2.1.2 Yeast 0-glucosidases 2.1.3 Mold 13-glucosidases 2.1.4 Plant (3-glucosidases 2.1.5 Animal /3-glucosidases 2.2 Mode of action of P-glucosidase
vii
(a) Glycosylation step (b) Deglycosylation step 2.3 Classification of I3-glucosidases 2.4 Crystal structure of 13-glucosidases
2.5 Chemical modification and site-directed mutagenesis for confirmation of amino acid residues in catalytic action of 13-glucosidases
2.6 Role of p-glucosidases, glycosyl transferases, Serine-aspartate repeat proteins in cell-surface associated phenomena
2.6.1 Role of 13-glucosidases in cell-surface-associated phenomena 2.6.2 Role of glycosyltransferases in cell-surface-associated phenomena 2.6.3 Role of serine-aspartate repeat (Sdr) protein in cell surface association 2.7 Applications of P-glucosidase
2.7.1 Applications based on hydrolytic activity 2.7.2 Applications based on synthetic activity 2.7.3 Other applications
3. MATERIALS AND METHODS 46
3.1 Strains, plasmids and growth conditions 3.1.1 Strains
3.1.2 Plasmids
3.1.3 Media and culture conditions 3.2 Molecular biological techniques
3.2.1 Preparation of genomic DNA library of P. etchellsii (a) Isolation of chromosomal DNA
(b) Isolation of plasmid and calf intestinal alkaline phosphatase treatment of the plasmid
(c) Partial digestion of Pichia chromosomal DNA and sizing of fragments
(d) Ligation and transformation in E. coli
(e) Screening for f3-glucosidase producing clones 3.2.2 Restriction analysis
3.2.3 Sequence analysis and delineation of correct ORF 3.2.4 PCR experiments
(a) For confirmation of mugA origin
(b) For introduction of desirable restriction sites flanking the mugA gene
3.2.5 Over-expression of mugA gene
(a) Cloning of PCR amplified fragment into pET29a vector (b) Induction studies on BL21 (DE3): pET29a clone 3.3 Sequence analysis and secondary structure prediction analysis
3.4 Localization of MUGA in recombinant E. coli pMG8: DH5a transformant:
preparation of sub-cellular Fractions i) Extracellular fraction
ii) Periplasmic fraction iii) Intracellular fraction 3.5 Purification of MUGA protein
3.5.1 Ammonium sulfate precipitation
3.5.2 Ion-exchange chromatography on DEAE-Sepharose 3.5.3 Hydroxyapatite chromatography
3.5.4 Gel filtration
3.6 Biochemical and kinetic characterization of purified MUGA 3.6.1 Determination of molecular mass and subunit composition 3.6.2 pH optimum and stability
3.6.3 Temperature optimum and thermal stability 3.6.4 Substrate specificity
3.6.5 Determination of kinetic parameters
3.6.6 Effect of metal ions, additives and n-alcohols 3.6.7 Far-UV CD spectrum of MUGA
3.7 Binding studies with MUGA
(i) Fluorescent microscopic experiment (ii) Competition experiment
ix
3.8 Analytical Methods 3.8.1 Enzyme assays
3.8.2 Soluble protein estimation (a) OD at 280 nm (b) Bradford's method
3.8.3 Polyacrylamide gel electrophoresis (a) SDS-PAGE
(b) Native - PAGE (c) PAGE - Zymogram 3.9 Equipment and chemicals
3.9.1 Equipment 3.9.2 Chemicals
4. RESULTS 80
4.1 Molecular cloning and characterization of mugA gene 4.1.1 Cloning of the novel (3
-
glucosidase-
like genefrom Pichia etchellsii
4.1.2 Characterization of mugA gene 4.2 Sequence analysis of MUGA protein 4.3 Secondary structure prediction
4.4 Purification and characterization of MUGA 4.4.1 Localization of MUGA
4.4.2 Purification of MUGA
4.4.3 Biochemical characterization of MUGA
4.4.3.1 Determination of molecular mass 4.4.3.2 pH optimum and stability
4.4.3.3 Temperature optimum and thermal stability 4.4.3.4 Substrate specificity
4.4.3.5 Determination of kinetic constants on MUG and pNPG
4.4.3.6 Effect of metal ions, additives and n-alcohols
4.4.3.7 Equilibrium CD spectrum studies 4.5 Binding of MUGA protein to yeast cells
4.6 Competition experiments
4.7 Over-expression of MUGA protein
5. DISCUSSION 128
6. CONCLUSIONS 141
REFERENCES 146
BIO-DATA OF AUTHOR
xi
