The Application of Hydrolytic Enzymes for
Biotransformations of Natural Products in NonAqueous Reaction Conditions
Submitted by
Joseph Bull
to the University of Exeter as a thesis for the degree of Doctor of Philosophy in
Biological Sciences, (June 2009).
This thesis is available for Library use on the understanding that it is copyright material
and that no quotation from the thesis may be published without proper
acknowledgement.
I certify that all material in this thesis which is not my own work has been identified and
that no material has previously been submitted and approved for the award of a degree
by this or any other University.
Abstract
Flavonoids are naturally occurring compounds that are consumed regularly in
the diet. The property of flavonoids, which they are most commonly known for, is their
antioxidant activity. Other potential pharmaceutical applications of flavonoids can be
related to their enzyme inhibition, anti-allergic, anti-inflammatory, anti-microbial and
anti-cancer properties. Lipases have been used effectively in the production of flavonoid
ester derivatives that have shown both increased antioxidant and antimicrobial activity.
Enzymatic esterifications of flavonoids are performed in organic solvents that increase
substrate solubility of complex organic molecules.
For the esterification of compounds in non-aqueous reaction conditions, vinyl
esters are often preferred as substrates compared to carboxylic acids (which can be
involved in reversible reactions, due to the formation of the by-product, water). In this
study a group of vinyl esters of tert-butoxycarbonylated amino acid derivatives were
synthesized to study alongside a range of commercially available vinyl esters.
The synthesis of ester derivatives of naringin using a range of hydrolytic
enzymes has been studied. A range of small to medium sized commercially available
vinyl esters (C2- C10), as well as amino acid vinyl esters were selected for the
biotransformations. For the esterification of naringin, small-scale reactions were carried
out for 72 hrs and the reaction mixtures were analysed by HPLC. Lipases from the
species Pseudomonas stutzeri, Candida antarctica and Alcaligenes spp. performed more
than 80% conversion of naringin with some of the selected acylating agents. Reactions
carried out with P. stutzeri lipase were scaled up to isolate the product of the
biotransformation. None of the screened enzymes were successful in the acylation of
naringin with the amino acid vinyl esters.
Assays were carried out to compare the antioxidant activity of naringin and the
synthesized derivatives. Two of the acyl derivatives showed a greater antioxidant
activity in the reduction of Cu2+, compared to naringin.
Aminoglycosides are antibiotics that have anti-bacterial properties. As well as
their anti-bacterial properties some have been employed for their ability to suppress stop
codons, which is a useful property in reducing symptoms of some hereditary disorders.
2
In the present work attempts were made to derivatise an aminoglycoside by
acylating it with the amino acid vinyl esters, using hydrolytic enzymes. Despite
screening with various proteases in different solvents, the acylation of amikacin was not
succesful during this investigation.
3
CONTENTS
Abstract .........................................................................................................................2
List of Tables ................................................................................................................9
List of Figures .............................................................................................................10
List of Appendices ......................................................................................................15
Acknowledgements .....................................................................................................16
Abbreviations .............................................................................................................17
Species Abbreviations ................................................................................................20
CHAPTER 1.Introduction .......... Error! Bookmark not defined.
1.1 Biocatalysis............................................ Error! Bookmark not defined.
1.1.1 Applications of Biocatalysis .................................. Error! Bookmark not defined.
1.1.2 Biocatalysis in Non-Aqueous Media .................... Error! Bookmark not defined.
1.1.3 Hydrolase Enzymes ............................................... Error! Bookmark not defined.
1.1.3.1 Lipases ................................................................. Error! Bookmark not defined.
1.1.3.2 Proteases.............................................................. Error! Bookmark not defined.
1.1.4 Biocatalysis in the Synthesis of Pharmaceutical CompoundsError! Bookmark
not defined.
1.2 Flavonoids ............................................. Error! Bookmark not defined.
1.2.1 Flavonoids .............................................................. Error! Bookmark not defined.
1.2.2 Structure of Flavonoids ........................................ Error! Bookmark not defined.
1.2.3 Naringin ................................................................. Error! Bookmark not defined.
1.2.4 Biotransformation of Flavonoids ......................... Error! Bookmark not defined.
1.2.5 Biotransformations of Naringin ........................... Error! Bookmark not defined.
1.3 Spinal Muscular Atrophy .................... Error! Bookmark not defined.
1.3.1 Spinal Muscular Atrophy ..................................... Error! Bookmark not defined.
1.3.2 SMN and Aminoglycosides ................................... Error! Bookmark not defined.
1.3.3 Biotransformation of Aminoglycosides ............... Error! Bookmark not defined.
1.4 Project Aims ......................................... Error! Bookmark not defined.
4
2. Amino Acid Vinyl Ester SynthesisError!
Bookmark
not
defined.
2.1 Introduction .......................................... Error! Bookmark not defined.
2.2 Synthesis ................................................ Error! Bookmark not defined.
2.2.1 Synthetic Routes .................................................... Error! Bookmark not defined.
2.2.1.1 Amine Protection ................................................ Error! Bookmark not defined.
2.2.1.2 Palladium Catalysed Transvinylation ReactionError! Bookmark not defined.
2.3 Results and Discussion ......................... Error! Bookmark not defined.
2.3.1 Amine Protection ................................................... Error! Bookmark not defined.
2.3.2 Palladium Catalysed Transvinylation Reaction . Error! Bookmark not defined.
2.3.2.1 Basic Polar Amino
Acids…………………………………….……….……..Error! Bookmark not defined.
2.3.2.1.1
Lysine…………………………………………………………………….....Error!
Bookmark not defined.
2.3.2.1.2
Histidine
and
Arginine
Derivatives………………………………………....Error! Bookmark not defined.
2.3.2.2 Non-Polar Amino Acid Esters
.................. ……………………………………………....Error! Bookmark not defined.
CHAPTER 3. ................................ Error! Bookmark not defined.
Enzymatic Acylations of NaringinError! Bookmark not defined.
3.1. Introduction ......................................... Error! Bookmark not defined.
3.2 Enzymatic Screening of Naringin DerivatisationError!
Bookmark
not defined.
3.2.1 Materials ................................................................ Error! Bookmark not defined.
3.2.1.1 Substrates ............................................................ Error! Bookmark not defined.
3.2.1.2 Enzymes .............................................................. Error! Bookmark not defined.
3.2.1.3 Reaction Media ................................................... Error! Bookmark not defined.
3.2.1.4 TLC ..................................................................... Error! Bookmark not defined.
3.2.1.5 HPLC................................................................... Error! Bookmark not defined.
3.2.2 Methods .............................................................. Error! Bookmark not defined.
5
3.2.2.1 Enzyme Screening for Acylations of Naringin Error! Bookmark not defined.
3.2.2.2 Protein Sequence Alignment ............................. Error! Bookmark not defined.
3.2.2.2 Protein Homology Modelling ............................ Error! Bookmark not defined.
3.3 Synthesis of Naringin Ester DerivativesError! Bookmark not defined.
3.3.1 Synthetic Route ..................................................... Error! Bookmark not defined.
3.3.1.1 Lipase-catalysed Reactions ................................ Error! Bookmark not defined.
3.4 Results and Discussion ......................... Error! Bookmark not defined.
3.4.1 Enzyme Screening ................................................. Error! Bookmark not defined.
3.4.1.1 Screening Conditions ......................................... Error! Bookmark not defined.
3.4.1.2 Aliphatic Vinyl Esters ........................................ Error! Bookmark not defined.
3.4.1.3 Aromatic Vinyl Esters .......................................... Error! Bookmark not defined.
3.4.1.4 Succinic Anhydride .............................................. Error! Bookmark not defined.
3.4.1.5 Amino Acid Vinyl Esters ..................................... Error! Bookmark not defined.
3.4.1.5.1
N-tert-Butoxycarbonyl-L-Phenylalanine
Vinyl
Ester……………………...Error! Bookmark not defined.
3.4.1.5.2 N-tert-Butoxycarbonyl-L-Valine and N, N-di-tert-Butoxycarbonyl-L-Lysine
Vinyl
Esters…………………………………………………………………………...Error!
Bookmark not defined.
3.4.2 Synthesis of Naringin Derivatives ........................ Error! Bookmark not defined.
3.4.2.1 Aliphatic Vinyl Esters ........................................ Error! Bookmark not defined.
3.4.2.2 Aromatic Vinyl Esters ....................................... Error! Bookmark not defined.
3.4.2.3 N-tert-Butoxycarbonyl-L-Phenylalanine Vinyl Ester ... Error! Bookmark not
defined.
3.4.4 P. stutzeri Lipase Structure .................................. Error! Bookmark not defined.
CHAPTER
4.
Antioxidant
Activity
of
Naringin
Derivatives.................................................................................Err
or! Bookmark not defined.
4.1
Introduction
......................................................................................Error! Bookmark not
defined.
6
4.2 Screening for Antioxidant Activity of Naringin Derivatives . Error!
Bookmark not defined.
4.2.1 Materials ................................................................ Error! Bookmark not defined.
4.2.2 Methods .................................................................. Error! Bookmark not defined.
4.2.2.1 Trolox Standard ................................................. Error! Bookmark not defined.
4.2.2.2 Antioxidant Sample ............................................ Error! Bookmark not defined.
4.2.2.3 Total Antioxidant Capacity Assay .................... Error! Bookmark not defined.
4.3 Results and Discussion ......................... Error! Bookmark not defined.
CHAPTER 5. Screening for Acylations of an Aminoglycoside
Derivative
with
Amino
Acid
Vinyl
Esters
........................... …………………Error! Bookmark not defined.
5.1 Introduction .......................................... Error! Bookmark not defined.
5.2 Synthesis ................................................ Error! Bookmark not defined.
5.2.1 Synthetic Routes .................................................... Error! Bookmark not defined.
5.2.1.1 Amine Protection ................................................. Error! Bookmark not defined.
5.3 Enzymatic Screening of Amikacin DerivatisationError!
Bookmark
not defined.
5.3.1 Materials ................................................................ Error! Bookmark not defined.
5.3.1.1 Substrates ............................................................. Error! Bookmark not defined.
5.3.1.2 Enzymes ............................................................... Error! Bookmark not defined.
5.3.1.3 Reaction Media .................................................... Error! Bookmark not defined.
5.3.1.4 TLC ...................................................................... Error! Bookmark not defined.
5.3.2 Methods .................................................................. Error! Bookmark not defined.
5.3.2.1 Non-Aqueous Protease-catalysed Reactions ........ Error! Bookmark not defined.
5.3.2.2 Aqueous Protease-catalysed Reactions ................ Error! Bookmark not defined.
5.4 Results and Discussion ......................... Error! Bookmark not defined.
5.4.1 Amine Protection ................................................... Error! Bookmark not defined.
5.4.2 Biotransformation Screening ............................... Error! Bookmark not defined.
5.4.2.1 Non-Aqueous Protease Biotransformation .......... Error! Bookmark not defined.
5.4.2.2 Aqueous Protease Biotransformation ................... Error! Bookmark not defined.
7
6. Experimental Details ............... Error! Bookmark not defined.
6.1 Synthesis of N, N-di-tert-Butoxycarbonyl-L-Lysine ......... Error! Bookmark not
defined.
6.2 Synthesis of N, N-di-tert-Butoxycarbonyl-L-Lysine Vinyl Ester. ........... Error!
Bookmark not defined.
6.3 Synthesis of N-tert-Butoxylcarbonyl-L-Phenylalanine ....... Error! Bookmark not
defined.
6.4 Synthesis of N-tert-Butoxylcarbonyl-L-Phenylalanine Vinyl Ester ........... Error!
Bookmark not defined.
6.5 Synthesis of N-tert-Butoxylcarbonyl-L-Valine ...... Error! Bookmark not defined.
6.6 Synthesis of N-tert-Butoxycarbonyl-L-Valine Vinyl Ester . Error! Bookmark not
defined.
6.7 Synthesis of Naringin-6’’-O-Acetate ...................... Error! Bookmark not defined.
6.8 Synthesis of Naringin-6’’-O-Benzoate .................... Error! Bookmark not defined.
6.9 Synthesis of Naringin-6’’-O-Butyrate .................... Error! Bookmark not defined.
6.10 Synthesis of Naringin-6’’-O-Cinnamate .............. Error! Bookmark not defined.
6.11 Synthesis of Naringin-6’’-O-Crotonate ................ Error! Bookmark not defined.
6.12 Synthesis of tetra-N-tert-Butoxcarbonyl AmikacinError! Bookmark not defined.
CHAPTER 7. Summary and Future Work.Error!
Bookmark
not defined.
7.1 Summary ............................................... Error! Bookmark not defined.
7.2 Future Work ......................................... Error! Bookmark not defined.
Appendix…………………………………………………………………………....145
References ....................................................................... Error! Bookmark not defined.
7
8
List of Tables
Table 3.1. Table showing the levels of conversion of naringin with acyl donors,
catalysed by lyophilised enzymes…................................................................................79
Table 3.2. Table showing the retention time of naringin and the lipase-catalysed
derivatives………………………………………………………………………………80
Table 3.3. Table showing the 13C chemical shift (ppm) for 6’’-CH2-OH of naringin and
its lipase-catalysed derivatives........................................................................................91
9
List of Figures
Figure 1.1. Esterification reaction mechanism occuring in the active site of lipase
enzymes……………………………………………………………………...................21
Figure 1.2. Structures of 6-O-acylated derivatives of glucopyranose, galactopyranose
and mannopyranose……………………………………………..…………………...…28
Figure 1.3. The acylation of digitonin.............................................................................29
10
Figure 1.4. Acetophenone derivatives deprotected by lipases PPL and CCL.................30
Figure 1.5. Scheme showing the enzymatic synthesis of vinyl uridine esters and
polymerisation.................................................................................................................31
Figure 1.6. Scheme showing the biocatalytic preparation of different doxorubicin
derivatives........................................................................................................................31
Figure 1.7. Scheme showing the acetylation of GD3 using subtilisin in the presence of
triethylamine....................................................................................................................33
Figure 1.8. A diagram showing NSAIDS that have been synthesized using a
biocatalyst........................................................................................................................34
Figure 1.9. Scheme showing the resolution of the racemic precursor azetidinone acetate
two different Pseudomonas species lipases.....................................................................34
Figure 1.10. Scheme showing two step enzymatic modification of paclitaxel using
thermolysin and CALB....................................................................................................35
Figure 1.11. A diagram showing the flavane structure of flavonoids.............................37
Figure 1.12. A diagram showing the structure of the flavonoid naringin.......................38
Figure 1.13. A diagram showing the preferred site of flavonoid glycosylation at the C’3
hydroxyl group of the B-ring...........................................................................................40
10
Figure 1.14. Scheme showing the glycosylation of rutin by a transglycosyaltion enzyme
from B. stearothermophilus.............................................................................................40
Figure 1.15. Scheme showing the lipase-catalysed acylation of isoquercitin.................43
Figure 1.16. Scheme showing the acylation of naringin with CALB in ionic
liquids..............................................................................................................................45
11
Figure 1.17. Structures of the aminoglycosides gentamicin, tobramycin and
amikacin………………………………………………………………………...............50
Figure 1.18. Scheme showing the biotransformation of amikacin with divinyl sebacate
using B. subtilis protease.................................................................................................51
Figure 2.1. Synthetic route for the tert-butoxcarbonyl derivatives of amino
acids.................................................................................................................................56
Figure 2.2. Synthesis of amino acid vinyl esters using N-tert-butoxycarbonyl-protected
amino acids as substrates.................................................................................................57
Figure 2.3. Reaction mechanism for the amine protection of L-lysine, L-phenylalanine
and L-valine using di-tert-butoxycarbonate....................................................................58
Figure 2.4. A diagram representing the mechanism for the transvinylation of amino acid
derivatives........................................................................................................................60
Figure 2.5. Reaction scheme for the synthesis of N, N-di-tert-butoxycarbonyl-L-lysine
vinyl ester........................................................................................................................61
Figure 2.6. Histidine derivatives selected for transvinylation.........................................62
Figure 2.7. Nucleophilic attack of the imidazole tert-butoxycarbonyl protection group
by a hydroxide ion...........................................................................................................63
Figure 2.8. Scheme showing anion exchange of acetate group of Pd(OAc)2 for the
histidine derivative..........................................................................................................64
Figure 2.9. Arginine derivatives selected for transvinylation.........................................64
Figure 2.10. The attempted synthesis of vinyl esters of histidine and arginine
derivatives........................................................................................................................65
12
Figure 2.11. The synthetic route for the synthesis of the vinyl esters of N-tertbutoxycarbonyl-L-phenylalanine and N-tert-butoxycarbonyl-L-valine..........................68
Figure 3.1. Formula used to calculate the levels of conversion of naringin with the acyl
donors to form the respective ester derivative.................................................................76
Figure 3.2. Scheme showing the synthesis of naringin ester derivatives using vinyl acids
as an acyl donor...............................................................................................................77
Figure 3.3 HPLC chromatograph showing multi-product formation catalysed by P.
stutzeri
lipase
reaction,
when
using
succinic
anhydride
as
an
acyl
donor................................................................................................................................83
Figure 3.4. HPLC chromatograph showing the control reaction for naringin and succinic
anhydride.........................................................................................................................84
Figure 3.5. Scheme showing the attempted synthesis of naringin derivatives with the Ntert-butoxycarbonyl-L-valine vinyl esters and N, N-di-tert-butoxycarbonyl-L-lysine
vinyl ester........................................................................................................................86
Figure 3.6. Reaction scheme showing the biotransformation of naringin with aliphatic
vinyl acid esters...............................................................................................................87
Figure. 3.7. Reaction scheme showing the biotransformation of naringin with aromatic
vinyl acid esters...............................................................................................................88
Figure 3.8. HPLC chromatograph of reaction between naringin and N-tertbutoxycarbonyl-L-phenylalanine vinyl ester, catalysed by Alcaligenes spp. lipase
A2....................................................................................................................................89
Figure 3.9. Scheme showing the attempted synthesis of the N-tert-butoxycarbonyl-Lphenylalanine naringin-ester............................................................................................89
13
Figure. 3.10. +NanoESI spectra of the reaction mixture from the attempted acylation of
naringin
with
the
vinyl
ester
N-tert-butoxycarbonyl-L-phenylalanine
vinyl
ester..................................................................................................................................90
Figure 3.11. Amino acid sequence of alignment of P. stutzeri lipase compared to three
lipases with similar sequence identity.............................................................................93
Figure 3.12. Modelled structure of the active site of P. stutzeri lipase, showing the
active site residues of the catalytic triad..........................................................................94
Figure 3.13. Cartoon of the P. mendonica lipase structure...........................................110
Figure. 3.14. The electrostatic potential surface calculated for the P. stutzeri lipase
model.............................................................................................................................111
Figure 4.1. Formula used to calculate naringin and its derivatives sample antioxidant
capacity..........................................................................................................................100
Figure 4.2. Diagram showing the structure of naringin and its derivatives complexing to
Cu2+ to reduce the copper ion to Cu+.............................................................................101
Figure 4.3. Diagram showing the free radical scavenging acivity of the 4-OH B-ring of
the flavonoid naringin....................................................................................................102
Figure 4.4. Structure of naringin flavane compared to that of a flavane structure, which
has been shown to provide maximum antioxidant activity...........................................102
Figure 4.5. Graph comparing the relative antioxidant capacity of naringin and its
derivatives......................................................................................................................104
Figure 5.1. Synthetic route for the tetra-N-tert-butoxcarbonyl derivatives of amikacin,
using di-tert- butyl dicarbonate.....................................................................................110
Figure 5.2. Reaction mechanism for the amino protection of amikacin using di-tertbutoxycarbonate to yield tetra-N-tert-butoxtycarbonyl amikacin.................................115
14
Figure 5.3. The attempted synthesis of amino acid ester derivatives of tetra-N-tertbutoxycarbonyl amikacin in non-aqueous and aqueous reaction media using
proteases........................................................................................................................118
List of Appendices
Appendix 1. Ramachadran Plot for P. stutzeri Lipase..................................................145
Appendix 2. Ramachadran Plot for P. mendonica Lipase (PDB code 2fx5)................146
Appendix 3. Trolox Concentration Standard Curve......................................................147
Appendix 4. Table showing the sample antioxidant activity capacity of naringin and its
derivatives......................................................................................................................148
15
Acknowledgements
I would like to thank my supervisors Prof. Jenny Littlechild and Dr. Steve
Taylor for all their help and supervision, as well as giving me the enthusiasm to meet
challenging topics head on. I would like to acknowledge the EPSRC for funding the
project and for paying me.
I would like to thank all the guys who have worked at the Biocat during my time
here (especially all the drinking buddies). A special mention goes to Dr. Kirsty Line for
helping me with my sequence alignment and modelling plus passing on all her HPLC
16
knowledge. Thanks also go to Dr. Steve Connelly for getting me started with my
Chemistry and Chris Sayer for helping me with my IRs.
In addition, my thanks go to members of the former Chemistry department, past
and present, for useful discussions on many of the chemical aspects of the project. In
particular I would like to thank Dr. Mark Wood for all his advice on amino acid
Chemistry. I appreciate all the help Gwen Batten and Nick Elliot, have given me in
locating the necessary apparatus required to complete my investigation, during the
restructuring of the department.
I would like to thank the guys at Microanalysis Ltd (Okehampton) for running
my elemental analysis samples. I would also like to express my gratitude to Dr. Paul
Gates (University of Bristol), for running all my mass spec. samples and helping me
with the analysis.
Finally I would like to thank my fiancée, Alex, for all the support she has given me and
for helping me to retain my sanity during the process of finishing this thesis.
Abbreviations
ACAT, Acyl coenzyme A cholesterol acyltransferase
Acyl CoA, Acyl coenzyme A
AOT, Sodium bis (2-ethylhexyl) sulphosuccinate
Å, Angstroms
aP, Area % of product (HPLC)
Arg, Arginine
aS, Area % of substrate (HPLC)
Asn, Asparagine
Asp, Aspartic acid
17
([bmim]BF4), 1-butyl-3-methylimidazolium tetrafluoroborate
([bmim]BF6), 1-butyl-3-methylimidazolium hexafluorophosphate
Boc, tert-butoxycarbonyl
c, Conversion (%)
°C, Celsius
Cys, Cysteine
C, Cytosine
CAL, Candida antarctica lipase
CALB, Candida antarctica lipase B
calc., Calculated
CCL, Candida cylindracea lipase
CHN, Elemental analysis
CH3, Methyl group
DAPI, 4',6-diamidino-2-phenylindole.
ddH2O, double distilled water
DMF, N,N-dimethylformamide
DMSO, Dimethyl sulfoxide
e-, Electron
eq., Equivalent
EC, Enzyme commission number
FITC, Fluorescein isothiocyanate.
FV-VΙΙ, Coagulation factor seven
g, Grams
Gln, Glutamine
Glu, Glutamic acid
17
His, Histidine
hrs, Hours
HPLC, High performance liquid chromatography
im, Imidazole
kb, Kilobases
LDL, Low-density lipoprotein
Lys, Lysine
M, Molar
Me4Si, tetramethylsilane
Met, Methionine
18
mins, Minutes
m/z, Mass-to-charge ratio
μ, Micron
μl, Microlitres
μm, Micromoles
mg, Milligrams
ml, Millilitres
mM, Millimolar
mRNA, Messenger ribonucleic acid
MRSA, Methicillin-resistant Staphylococcus aureus
nm, Nanometres
nmol, Nanomole
NSAIDS, Non-steroidal anti-inflammatory drugs
Pbf, 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
PDB, Protein Database
Pd(OAc)2, Palladium (II) acetate
pKa, Acid dissociation constant
pH, Measure of acidity
Phe, Phenylalanine
ppm, Parts per million
Q, Glutamine
rpm, Revolutions per minute
rRNA, Ribosomal ribonucleic acid
SC, Subtilisin Carlsberg
Ser, Serine
SMA, Spinal muscular atrophy
SMN, Survival motor neurone
snRNPs, Small nuclear ribonucleoproteins
spp., Species
SOD, Superoxide dismutase
SR proteins, Serine and arginine-rich proteins
T, Thymine
TEAC, Trolox equivalence antioxidant capacity
tert, Tertiary
THF, Tetrahydrofuran
19
TLC, Thin layer chromatography
Tosyl, p-toluenesulfonate
tRNA, Transfer ribonucleic acid
TRITC, Tetramethylrhodamine isothiocyanate
UV, Ultra violet
Val, Valine
v/v, volume/volume
VRE, Vancomycin resistant Enterococci
w/v, weight/volume
Species Abbreviations
A. melleus, Aspergillus melleus
A. oryzae, Aspergillus oryzae
B. lentus, Bacillus lentus
B. stearothermophilus, Bacillus stearothermophilus
B. subtilis, Bacillus subtilis
Can. antarctica, Candida antarctica
Can. cylindracea, Candida cylindracea
Car. papaya, Carica papaya
E. coli, Escherichia coli
M. miehei, Mucor miehei
20
P. aeruginosa, Pseudomonas aeruginosa
P. cepacia, Pseudomonas cepacia
P. mendonica, Pseudomonas medonica
P. stutzeri, Pseudomonas stutzeri
R. niveus, Rhizopus niveus
R. oryzae, Rhizopus oryzae
Sta. aureus, Staphylococcus aureus
Str. griseus, Streptomyces griseus
21