Investigations on cellular nanoparticles required for synthesis of chitin the precursor for chitosan [Elektronische Ressource] / vorgelegt von Mayur Kumar Kajla

Mayur Kumar Kajla

Sujets

• Biologie

Informations

• Publié par: universitat_osnabruck
• Publié le: 01 janvier 2005
• Nombre de lectures: 41
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Investigations on cellular
nanoparticles required for synthesis
of chitin the precursor for chitosan



Dissertation

zur Erlangung des Grades eines Doktors
der Naturwissenschaften (Dr. rer. nat.)
an der Universität Osnabrück


vorgelegt
von Mayur Kumar Kajla
aus Himachal Pradesh, Indien

2 0 0 5 September
ii


























1 .Berichterstatter : Prof. Dr. H. Schrempf

2. Berichterstatter : Prof. Dr. Jürgen Hofemeister

iii
Index P age

1.0 Introduction 1
2.0 Material and methods 7
2.1 Materials 7
2.1 Media 7
2.1.1 Yeast peptone adenine dextrose (YPAD) 7
2 Yeast malt extract (YM) 7
3 Luria Burtani (LB) 8
4 M9 Minimal Medium 8
2.2 Buffers and stains 9
2.2.1 Standard buffers 9
2 Western transfer buffer 9
3 Acetonitrile buffer 9
4 Buffers for blue native PAGE 10
5 Protein loading buffer for SDS-PAGE 10
6 Coomassie staining solution 10
7 Silver and zinc staining of polyacrylamide gels 11
7.1 Silver staining 11
2 Zinc staining 12
2.3 E. coli and S. cerevisiae strains 12
2.4 Antibodies 13
2.5 Biochemical methods 14
2.5.1 Cultivation of yeast 14
2 Chitin synthase assay 14
2.1 WGA based assay 14
iv
2 CHB1 based assay 16
2.6 Isolation and purification of chitosomes 17
2.7 Separation of the proteins 20
2.7.1 SDS-PAGE 20
2 2-dimensional blue native PAGE 20
2.8 Western blotting 22
2.9 Isolation and purification of chitin binding protein 23
3.0 Differential extraction of the proteins 25
3.1 Determination of protein concentration 26
3.2 Purification of proteins via HPLC and gel filtration 26
3.3 Electron microscopy analysis of chitosomes 26
3.4 Additional enzymatic assays 28
3.4.1 UDP-N-acetyl-glucosamine pyrophosphorylase assay 28
3.4.2 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assay 28
3.4.3 Enolase assay 28
3.4.4 Pyruvate kinase assay 28
3.4.5 Pyruvate decarboxylase assay 29
3.5 Sample preparation for MALDI-MS analysis 29
4.0 Results 31
4.1 Identification and purification of chitosomal complexes 31
4.2 CHB1 as a tool for chitosomal quality control 37
4.3 Structural analysis of complexes via electron microscopy 39
4.4 Evaluation of different protein enrichment conditions 41
4.5 Identification of major proteins in chitosomal fractions 44
4.6 Characterization of identified proteins 46
4.7 Studies on CSI-HA strain 56
5.0 Discussion 60
v
6.0 Summary 72
7.0 Bibliography 73

Affidavit
Acknowledgements



















vi
List of figures and tables Page

Fig. 4.1 Purification of chitosomes from wild type strain 32
Fig. 4.2 Purification of chitosomes frochsm3 mutant 34
Fig. 4.3 Comparison of the CSI activity assays 36
Fig. 4.4 Purification of chitin binding protein CHB1 38
Fig. 4.5 EM examination of chitosomal complexes 41
Fig. 4.6 Enrichment of chitosomal proteins using salts 43
Fig. 4.7 Separation of proteins via 2-D PAGE 45
Fig. 4.8 Clustal W alignment of N-termini of two proteins 47
Table 4.9 (i) List of peptide masses 48
Table 4.9 (ii) List of peptide masses 49
Table 4.10 Summary of the identified proteins 50
Fig. 4.11 and 4.12 Evaluation of enzymatic activities 52, 53
Fig. 4.13 Immunodetection of proteins in chitosomal fractions 54
Fig. 4.14 Immunodetection of GAPDH and Pdc 55
Fig. 4.15 Analysis of CSI-HA tagged protein 57
Fig. 4.16 Purification of chitosomes from CSI-HA strain 58
Fig. 4.17 Detection of CSI-HA in chitosomal fractions 59


vii
Abbreviations used in the text

ACN Acetonitrile
ADH Alcohol dehydrogenase
AGM Angewandte genetic der mikroorganismen
APS Ammonium persulphate
A600 Absorbance at 600 nm
BSA Bovine serum albumin
0C Degree celcius
Ca C irca
CAPS 3-c[yclohexylamino]-1-propanesulfonic acid
CHB1 Ch itin binding protein 1
cm C entimeter
CSI C hitin synthase I
CSII C hitin synthase I I
CSIII C hitin synthase III
1D One dimensional
DMSO D imethyl sulphoxide
E. coli Escherichia coli
EDTA Ethylene diamine tetra-chloro acetic acid
Eno Enolase
FAS Fatty acid synthase
FC Folin-C iocalteu reagent
FKS Glucan synthase
g Gram/s
GAPDH Glyceraldehyde 3- phosphate dehydrogenase
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GdHCl Guanidine hydrochloride
GlcNAc N-acetyl-D-glucosamine
HA H aemagglutinin
HPLC H igh pressure liquid chromatography
HRP H orseradish peroxidase
Hrs H ours
IPTG Isopropyl-thio-galactoside
kDa K ilo Da lton
LB Luria-Burtani
M M olar
mg M illigram
µl M icroliter
ml M illiliter
mM M illimolar
β-ME β-mercaptoethanol
MALDI-MS M atrix assisted laser desorption ionization mass spectrometry
Min Min utes
MW M olecular weight
N Normal
NADH Nicotinamide adenine dinucleotide hydroxide
NCBI National center for biotechnology information
Ni-NTA Ni-nitrilotriacetic acid
nm Nanometer/s
ORF Open reading frame
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
Pdc Pyruvate decarboxylase
ix
PEP Phosphoenol pyruvate
Ppm Parts per million
PVDF Polyvinylidene fluoride
Pyk Pyruvate kinase
rpm Rotations per minute
S. cerevisiae Saccharomyces cerevisiae
SDS Sodium dodecyl sulphate
Sec Seconds
SGD Saccharomyces genome database
TEMED N, N, N’, Nt’e-tramethylethylenediamine
TFA Trifluoroacetic acid
TMB Tetra m ethyl benzidine
UDP Uridine-di-phosphate
UDP-GlcNAc Uridine-diphosphate-N-acetyl-D-glucosamine
U/ml Units per milliliter
UTP Uridine triphosphate
V Volts
WGA Wheat germ agglutinin
YM Yeast malt extract
YPAD Yeast peptone adenine dextrose






1
1.0 Introduction

Chitin is the second most abundant biopolymer presienn tn ature. It is an
unbranched polysaccharide of β(1-4) linNk-edac etyl, D-glucosamine units. As
much as 10 giga tones of chitin is produced and degrady emdi crob organisms each
year. Chitin is present in the cell walls of fungi,k elextosns of insects and
crustaceans. It plays an important role in morphogenesis suanrvdiv al of the
organisms. In addition to the protective covering thatnt aimnais structure, it also
serves as a dynamic interphase between cytosol and ther e enovuironment which
allows changes during growth and differentiation.
InS . cerevisia echitin is an important component of the cell walhl e and t
septum, a structure that separates the mother cell fromd authge hter. Within the
cell wall, chitin cross-links with glucan (Kapett eya.nl, 1999; Cabib and Duran,
2005). Glucan comprises a major portion (about 60%) of t hcee lly ewasaltl. Most of
it is β(1-3) glucan and gives rigidity to the cel l swmall. aAmount of β(1-6)
glucan is present interspersed among glucan-protein net. woTrkhe outer side of the
cell wall consists of a highly glycosylated layer of mopanronteins (see pictorial
diagram, page 2). Chitin is highly concentrated at otfh et hsei teprimary septum
where it is deposited as ring at the site of inbcuipdi. entT his ring remains in the
neck during bud emergence and growth. Once the bud qhuasire dac sufficient
growth, the plasma membrane invaginates and the disk itoinf cish deposited in the
neck. The secondary septum is then formed around boetsh osfi dthe disk forming
a trilayered septum. Later after the septation is completeh,it inc is deposited in the
lateral walls of the bud. Though present in small am-ou3%n)t ,( 1its presence in the
cell wall is vital for growth and survival in yeast e(tS hawa.l, 1991). In coordination
with the other components, chitin provides rigidit yt hteo cell wall and maintains