Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal...
Transcript of Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal...
![Page 1: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/1.jpg)
Biocatalysis at KTHKarl HultSchool of BiotechnologyRoyal Institute of Technology (KTH)Stockholm, Sweden
![Page 2: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/2.jpg)
Biocatalysis at KTHKarl HultSchool of BiotechnologyRoyal Institute of Technology (KTH)Stockholm, Sweden
To advance the understanding and use of enzyme catalysis
based on a broad interdisciplinary competence in enzymology, chemistry, molecular biology, and
computational chemistry
Vision
![Page 3: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/3.jpg)
Enzyme systems of special interests of the Biocatalysis Group
• Lipases with special interest in Candida antarctica lipase BSubstrate specificityReaction specificityUse in synthesis
• TransaminasesProduction of chiral amines from ketones
![Page 4: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/4.jpg)
Reaction mechanism of lipasesAcylation Deacylation
Acyl-enzyme
Ser105
His224
His224
His224
Oxyanion holeO
O NHN
O
NHHN
NHN
HN
HNHO
Gln106
Thr40
Ser105
HO
O
R*
R''
O
O
R*
R''O
Ser105
O
HO
R*
R''
O
Asp187
O
OAsp187
O
OAsp187
Ser105
His224
His224
His224
Oxyanion holeO
O NHN
O
NHHN
NHN
HN
HNHO
Gln106
Thr40
Ser105
HO
O
R'
R''
O
O
R'
R''O
Ser105
O
HO
R'
R''
O
Asp187
O
OAsp187
O
OAsp187
![Page 5: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/5.jpg)
Lipase B from Candida antarcticais a serine hydrolase belonging to the α/βhydrolase family with a central β-sheetSurrounded by α-helixes
Lipase B from Candida antarctica is very active and stable in most organic solvents
Candida antarctica lipase BCandida antarctica lipase B
The activesite is deep in theproteinstructure
![Page 6: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/6.jpg)
Lipase catalysed kinetic resolution of a chiral alcohol
OHROR
OHR
R'
O
O O
O
R'
OR
O
R'
OEnz
O
R'OHEnz
![Page 7: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/7.jpg)
Thr40
Acyl chain
Large groupof alcohol
Medium groupof alcohol
Stereoselectivity pocket
Leu278
Trp104
Ala281
Ile285
His224
Ser105
R-3-Hexyl octanoatein the active site of CALB wt
The size of the stereoselectivitypocket is set by Trp104
![Page 8: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/8.jpg)
R-3-Hexyl octanoatein the active site of CALB W104A
New volume created by W104A mutation
Ala104
Ser105His224
![Page 9: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/9.jpg)
Enantioselectivity changed 8 300 000 by one point mutation, ΔΔG# 40 kJ/mol.
kcat for the S-enantiomer increased 65 000 times, ΔΔG# 28 kJ/mol
OHThe enantioselectivity changed8 300 000 times by one point mutationafter rational design
Magnusson A, Hamberg A, Takwa M and Hult K, 2005, Angewandte Chemie Int Ed, 44: 4582
Enzyme Enan- KM kcat kcat/KM E tiomer mM s-1 M-1s-1 wt R 61 570 9400 1300000 S 71 0.00053 0.007 W104A R 29 4.4 150 0.15 S 34 34 1000
![Page 10: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/10.jpg)
-40
-20
0
20
40
kJ/m
ol ΔΔGΔΔH -TΔΔS
Enthalpic and entropic contributions to enantioselectivity for CALB W104A
R
S
OH OH OH OH OH
Vallin M, Syrén PO and Hult K, 2010 ChemBioChem 11: 411-416
OH
*
* Butyrate
17 4813 36 100
8
![Page 11: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/11.jpg)
Oxyanion hole Candida antarctica lipase B
Gln106
Thr40
R-2-Butyl octanoatetetrahedral intermediate in the active site of CALB
![Page 12: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/12.jpg)
Oxyanion hole details in Candida antarctica lipase B
O C O-HN NH
O-
O
HO
HN
Ser105OAsp187
His224
+HN
Thr40
Gln106
O C O-HN NH
O-
O
ZHHN
Ser105OAsp187
His224
+HN
Thr40Val
Gln106
Mutation Thr40Ala decreases the activity 2500 times, ΔΔG‡ = 20 kJ/mol
Substrate-assisted catalysis can rescue part of the lost of activityΔΔG‡ = 9 kJ/mol
Magnusson, Hult and Holmquist, 2001, JACS 123, 4354
![Page 13: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/13.jpg)
Substrate assisted catalysis can be used toincrease the yield of monoesters of diols
Patent application with BASF
![Page 14: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/14.jpg)
CALB catalyzes Michael additions
HO
NHO
NH
R'NuH
His
R''
NuH Michael addition
R'SH, R'NHR'', R'CHR''R''' or H2O2
![Page 15: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/15.jpg)
Reaction mechanism for Michael-type addition in CALB Ser105Ala with a thiol
Two-step mechanism suggested by abinitio calculations.
Computed potential energy surface B3LYP/6-31+G* energies (dashed line) relative the reaction complex (1). Solid line represents energies corrected for solvation effects.
O
HHH
H
N
N
H
SH
2TS
O
H
N
N
H
HH
SH
H
N
N
H
H
O
HHH
SH
4TS
O
HHH
S
N
N
H
HH
δ
δ
δ
δ
1 3
N
N
H
OH
H
SH
H
5
2TS 4TS1 3 5
0
10
-10
-20
ΔE /kcal/mol
0.02.0
-13.5
-1.7
-14.1
-15.7-21.2
0.7
-5.6
Reaction Coordinate
Carlqvist, P.; Svedendahl, M.; Branneby, C.; Hult, K.; Brinck, T.; Berglund, P. ChemBioChem, 2005, 6, 331-336.
![Page 16: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/16.jpg)
Diethyl amine reacts very fast with the wild type lipase
lipase
organic solventNH
O
O N
O
O
kappcat 810 min-1
Carlqvist, Svedendahl, Branneby, Hult, Brinck, Berglund, ChemBioChem, 2005, 6, 331-336.
![Page 17: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/17.jpg)
0
20
40
60
80
100
120
0 2 4 6 8 10
Time / min
Prod
uct f
orm
atio
n / %
CALB Ser105AlaCALB wild-typeCarrier without enzymeSubstrates only
Mutant-catalyzed Michael addition of acetyl acetone to methyl vinyl ketone is
extremely fast
kcat,app = 4 000 s-1
Svedendahl, M.; Hult, K.; Berglund, P. JACS 2005, 127, 17988
OCandida antarctica lipase B
Ser105Ala
1:1.4, bulk ,20 °C
O
O
O
O
O
![Page 18: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/18.jpg)
Summary of reactions and rates
Bond Rate (“kcat”) and reaction type min-1
“Natural” C-O transacylation 34 000
“Unnatural” C-C aldolase 0.045 C-O epoxidation 0.9 C-S Michael addition 4.3 C-N Michael addition 810 C-C Michael addition 240 000
![Page 19: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/19.jpg)
Enzyme contributes to catalysis by decreasing transition state energy
Enzyme + substrate
ΔΔG#, enzyme contribution
Transition state, no enzyme
ΔG#enz
Enzyme substratecomplex
Transition statewith enzyme
"KM"
"kcat/KM"
ΔG#no enz
"knon""(kcat/KM)/knon"
Free
ene
rgy,
G
Reaction coordinate
![Page 20: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/20.jpg)
How well is the tentative reactionmechanism using the active site?
Reaction ΔΔG‡ H-bond [kJ mol-1] equivalents
Epoxidation 39 2.0 Michael addition, C-S 42 2.2 Michael addition, C-C 48 2.5
The mutation Thr40Ala showed that one hydrogen bond in the oxyanion hole was worth almost 20 kJ mol-1 . This allows an estimation of the number of hydrogen bonds formed in transition state. The tentative reaction mechanism involves four hydrogen bonds.
![Page 21: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/21.jpg)
Polycondensation with acid end-capping functionalization
B BB B B BA A A A A AB BA A
AA
Catalyst
AAAA
AA
B BB B
B BB B
CACO
OBO OBO
Lipasen RO2CACO2R + (n+1) HOBOH + 2 R'O2CZ
CZO
+ 2n ROH + 2 R'OHO
nZC
O
![Page 22: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/22.jpg)
E‐OHE‐OOC(AB)nOOCZE‐OOC(AB)nOHE‐OOC(AB)nACOOR
E‐OOCZ
R’OOCZR’OH
ZCOOB(AB)n+xOOCZHOB(AB)n+xOOCZHOB(AB)n+xOHROOC(AB)n+xOHROOC(AB)n+xACOORYOOC(AB)m+nACOORROOC(AB)n+xOOCZROOCACOOR
ZCOOB(AB)xOHHOB(AB)xOHROOC(AB)xOHHOBOHROH
ZCOOB(AB)mOHHOB(AB)mOHROOC(AB)mOHHOBOHROH
ZCOOB(AB)m+nOOCZHOB(AB)m+nOHHOB(AB)m+nACOORHOB(AB)m+nOOCZROOC(AB)m+nACOORROOC(AB)m+nOOCZ
ZCOOB(AB)mOOCZHOB(AB)mOOCZROOC(AB)mOOCZ
HOB(AB)mOOCZHOB(AB)mOHROOC(AB)mOH
Cyclic product
Product:ZCOOB(AB)pOOCZ
Substrates:ROOCACOORHOBOHZCOOR’
B BB B B BA A A A A AB BA A
AA
Catalyst
AAAA
AA
B BB B
B BB B
22
H2O excluded
![Page 23: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/23.jpg)
23
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
1500
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
15000
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
1500
MALDI-TOF-MS kinetic study
Dynamic reaction system
![Page 24: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/24.jpg)
NMR of a one step CALB synthesized
telechelic polycondensation polymer
OO
O
OSH
HS
UV induced cross linking affords films
![Page 25: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/25.jpg)
OO
OO
O
O
O
O
O
Om n
10 10
OO
OO
O
O
O
O
O
O
O
On
10
n
OO
O
O
HS SH
7
10
n
OO
O
O
HS
Examples of polycondensation products
Examples of ring opening products
Other polymer products
Materials can be made from one-step synthesiswithout product purificationby UV initiated cross linking
![Page 26: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/26.jpg)
![Page 27: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/27.jpg)
![Page 28: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/28.jpg)
OO
O
O
O
RO
O
O
O
R-OH+ H
OO
O
O
O
RO
O
O
O
HOO
O
O
O
RO
O
O
O
H+
CALB
nn
CALB
Propagation
Initiation
Enzyme catalyzed polylactide synthesis is problematic due to enzymes’ substrate specificity
![Page 29: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/29.jpg)
NNHO
HO
Ser105
OH
O
O
H
H
H
N
O
N
His224
Asp187
O
Gln106
Thr40
O
OO
O
OO
Acyl donor
Acyl acceptor
Intermediate used for molecular modelling
![Page 30: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/30.jpg)
Important residues for mutation were identified by molecular modelling
Enzyme Initiation Propagation Rate (s-1) WT 40 1 Q157A 180 93 Q157A, I189A, L278A 770 83
![Page 31: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/31.jpg)
VINNOVA projectPer Berglund
Rational design of ω-transaminase from Arthrobacter citreus
• Aim: Increase the enantioselectivity for phenylacetone-type of substrates (98% ee)
• Homology model needed• Docking of pro-S and pro-R quinonoid
NH2 OF O F
NH2
![Page 32: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/32.jpg)
Rational design of ω-transaminase from Arthrobacter citreus
• Homology model needed
![Page 33: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/33.jpg)
Rational design of ω-transaminase from Arthrobacter citreus
Docking of pro-S and pro-R quinonoid
![Page 34: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/34.jpg)
Rational design of ω-transaminase from Arthrobacter citreus
![Page 35: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/35.jpg)
CNB05-01 or variantsNH2 O
F O FNH2
Mutant ee Enantiomer Enantiomeric % ratio Wt 98 S 100 Tyr331Cys 99,5 S 400 Val328Ala 58 R 4
![Page 36: Biocatalysis at KTH - Greenchem · Biocatalysis at KTH Karl Hult School of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden To advance the understanding and use](https://reader030.fdocuments.us/reader030/viewer/2022040710/5e10d30eb9dd9735cf19598c/html5/thumbnails/36.jpg)
Many good scientist students and friends have contributed to this work but most of all members of the Biocat and BioPol Groups at KTH