I!L!
Biotechnology!
Enzymreaktionen mit Milchinhaltsstoffen:
Was ist machbar?
Lutz Fischer
Institute of Food Science and Biotechnology
Department of BIOTECHNOLOGY
University of Hohenheim
STUTTGART, Germany
©Prof.
Dr. Lutz Fischer
I!L!
Biotechnology! Enzyme Technology in 2012
FOOD:
Process Conditions
sophisticated
mixture of ingredients
Screening of Desired Enzymes
- from -20°C up to +100°C
- aqueous pH 2-11
- various ionic strengths
- non-aqueous systems
- wild type (selektive media --> inducers)
- bioinformatics (data banks, sequence homology)
- DNA / RNA extraction (PCR, Metagenomis)
-  high throughput screening (robots, µL-scale)
-  patenting
Enzyme Production
Enzyme Design
-  recombinant hosts
prokaryotic, eukaryotic
-  efficient vector/host systems
-  secretion systems
- rational
(site-directed mutagenesis)
- evolutive
(in vitro random mutagenesis)
- biochemical (derivatisation)
Metabolic Engineering
- analysis of metabolic flux in cells
-  find bottle necks / close „wrong“ ways
-  genetic modification/engineering
©Prof.
Dr. Lutz Fischer
target:
selective
modification
I!L!
Biotechnology!
Data Content: BRENDA*
*http://www.brenda-enzymes.info
©Prof.
Dr. Lutz Fischer
I!L!
Biotechnology! Established Enzyme: β-Galactosidase
Application of Lactose Hydrolysis
Lactose
(Gal-β1,4-Glu)
Galactose
+
Glucose
©Prof.
Dr. Lutz Fischer
I!L!
Biotechnology!
Prominent β-galactosidases
β-galactosidases (EC 3.2.1.23): other name lactase,
"Lactase" (EC 3.2.1.108): enzyme from intestinal mucosa !
Microorganism
Fungi
Aspergillus niger
Aspergillus oryzae
extracellular
Yeast
Kluyveromyces lactis
Kluyveromyces fragilis
intracellular
Bacteria
Escherichia coli
Lactobacillus thermophilus
Leuconostoc citrovorum
Bacillus circulans
intracellular
# dependent
©Prof.
localized
Dr. Lutz Fischer
(extracellular)
pH optimum#
T-optimum
(°C)
3.0 - 4.0
5,0
55 - 60
50 - 55
6.5 - 7.0
6.6
30 - 35
30 - 35
7.2
6.2
6.5
6.0
40
55
65
65
Maxilact®
GODO-YNL2
Lactozym®
on strain
Mlichová, Z. & Rosenberg, M. (2006) J. Food Nutr. Res. 45
I!L!
Biotechnology!
Properties of β-galactosidases
Escherichia
coli
Molecular weight (kDa)
464
201
109-112
pI
4.6
5.1
4.6
pH optimum
7.2-7.4
6.2-6.4
2.5-4.0
pH stability
6-8
6.5-7.5
2-8
kcat ONPG sec-1
1.38 x 106
3.41 x 103
2.19 x 105
kcat lactose sec-1
5.10 x 103
1.55 x 103
1.91 x 105
Km ONPG (mM)
0.16
2.72
2.22
Km lactose (mM)
1.9
13.9
85-125
2.7 x 103
1.1 x 102
1.9 x 103
21
27.7
4
Na+, Mg2+
K+, Mg2+, Mn2+
None
kcat / Km lactose sec-1 mM-1
Ki galactose (mM)
Activators
©Prof.
Kluyveromyces Aspergillus
fragilis
niger
Dr. Lutz Fischer
Mahoney, R. R. (2003) Handbook of Food Enzymology, Chapter 65
I!L!
Biotechnology!
Screening Strategy on „Molecular Level“
enzymes from non-culturable microorganisms
Metagenome*
*Uchyama and Miyazaki, 2009, Curr. Opin. Biotechnol., Vol. 20, 616
©Prof.
Dr. Lutz Fischer
BRAIN AG, Zwingenberg, D
I!L!
Biotechnology! Metagenome-Screening: β-Galactosidases*
X-Gal agar plates
microtiter plates
1. screening level
2. screening level
metagenomic library
in E. coli on
LB-Agar-Platten
enzyme activity with
lactose as substrate
double layer agar
(X-Gal)
milk
*Karin Jaindl, Sarah Gulan, in preparation, University of Hohenheim, project partner BRAIN AG
©Prof. Dr. Lutz Fischer
I!L!
Biotechnology!
Hydrolysis of Lactose in Milk
- 500 mL milk at 8°C
-  comparison with commercial β-galactosidase „GODO“ (K. lactis)
©Prof.
-  analysis by HPLC (accuracy down to 0,01 g/L)
Dr. Lutz Fischer
I!L!
Biotechnology! Performance in lactose hydrolysis
•  Overview of the metagenome candidates and Godo-YNL2
à usage of 0.83 nkatLactose, 8°C / mL milk
50
lactose-concentration [g/L]
45
M1
40
M2
35
M3
30
M4
25
M5
20
M6
15
M7
10
Godo
5
0
0 50 100 150 time [h]
©Prof.
Dr. Lutz Fischer
200 250 300 I!L!
Biotechnology!
Performance of the best candidate
50
1
0,9
45
lactose concentration (g/L)
0,8
0,7
40
0,6
0,5
35
0,4
0,3
0,2
30
0,1 gL-1
0,1
0
25
0
10
20
30
40
50
60
20
15
GODO (K. lactis)
10
Metagenome-Enzyme*
5
0
0
10
20
30
40
50
60
time [h]
©Prof.
Dr. Lutz Fischer
*Sarah Gulan, Nico Böhmer in preparation, University of Hohenheim
I!L!
Biotechnology!
Immobilized β-Galactosidases
SEPABEADS® EC-HFA: enzyme carrier hetero-functional amino
•  matrix
•  functional group
•  particle size
•  pore diameter
polymethacrylate
amino epoxide
100 – 300 µm
10 ‐ 20 nm
•  >90% activity yield after
immobilization*
*Simone Ritter (2011) Diploma Thesis
Dr. Lutz Fischer
©Prof.
www.resindion.com
I!L!
Biotechnology!Continuous
hydrolysis of lactose in milk
4 °C, fresh milk (1,5 % fat), flow rate 60 mL/h, 216 µkatLactose,4°C,
column volume 270 mL
Time
[days]
Lactose
[g/L]
Degree of hydrolysis
[%]
1
0,050 ± 0,00
99,9
2
0,049 ± 0,01
99,9
3
0,060 ± 0,00
99,9
4
0,072 ± 0,01
99,8
5
0,079 ± 0,01
99,8
6
0,19 ± 0,00
99,6
7
0,41 ± 0,00
99,1
14
1,50 ± 0,01
96,7
21
1,62 ± 0,03
96,5
85% residual activity after 21 days
©Prof.
Dr. Lutz Fischer
*Simone Ritter (2011) Diploma Thesis
I!L!
Biotechnology!
Beispiel: Zufallsmutagenese
Cold adaptation of serine-protease*
Aktivität
bei 10°C
Bezeichnungen sind hier
Abkürzungen für Varianten,
keine Aminosäureaustausche
©Prof.
Dr. Lutz Fischer
*Wintrode et al. (2000) Biol. Chem. 275 (41), 31635-40
6,6-fach schneller !!!
*Wintrode et al. (2000) Biol. Chem. 275 (41), 31635-40
I!L!
Biotechnology!
Catalysis on lactose
enzymatic
hydrolysis
Lactose-free milk,
whey products
- Increased digestibility,
sweetness, solubility
trans
galactosylation
Galactosyl oligosaccharides (GOS)
Lactose
(Gal-Glu)
- Increased solubility
- Prebiotic (baby foods)
- Laxative
enzymatic
chemical
isomerisation
catalysis
Lactulose (Gal-Fru)
Vivinal® GOS
- Most popular laxative
- 10-times value of lactose
- Prebiotic (FOSHU list)
- Baby foods as bifidogenic factor
- Increased sweetness, solubility
©Prof.
Dr. Lutz Fischer
Harju, M. (2001) Int. J. Dairy Technol. 54
I!L!
Biotechnology!
HOCH2
HO
O
OH
Synthesen mit Hydrolasen
HOCH2
R1
REVERSE
HYDROLYSE
Allgemeiner
HOCH
22
O HOCH
Glycosidase
OH
OH
OH
HO
OH
OH
HO
HO
O
O
Glycosidase
Reaktionsweg
OH
OH
TRANSO
R
OH
HO
OH
HOGLYCOSYLIERUNG
O
R1
1
O
O
HOCH2
HO
+ R2OH
(Nukleophil)
Glycosidase
O
OH
HOCH2
OH
O
O
HO
Glycosidase
O
OH
©Prof.
Dr. Lutz Fischer
Glycosidase
Glycosidase
+
OH
OH
R2
OH
O
OH
R OH
H2O
O
-- H
2
O
HOCH2
HO
+
1
Glycosidase
Glycosidase
OH
OH
- R2OH
(Nukleophil)
HOCH2
++
O
O
H2O
O
++ H
2
HOCH2
*
OH
**
+
+
R1OH
R1OH
Glycosidase
I!L!
Biotechnology!
…bereits in den 90er Jahren
OH
Cl
D-Panthenol
H
HO
(35%)
OH H
N
O
OH (100%)
*
Chlorphenisin
OH
40°C, 20% DMF oder
(65%)
O
Aspergillus oryzae
Acetonitril, 2-4h, 5-6 gl-1 (TG)
Thermomyces
lanuginosus
40°C, 72h (TG)
OHOH
O
H O
H
HO
H
HN
OH
H
H
OH
OH
10
COOH
12-OH-Laurinsäure
20% Acetonitril, 4h, 1 gl-1 (TG)
H
OH
H
OH
tert-Amylalkohol
10
(0,3 gl-1 )
OH
O
OH
sek.-Esteralkohol (8,3 gl-1 )
OH
OH
O
O
Dr. Lutz Fischer
OH
Pyrococcus furiosus
75-95°C, 15-17h, (TG)
1-Dodecanol (1,8 gl-1 )
©Prof.
OH
Mandel (Immobilisat), 37°C,
ß-D-Glucose
OH
(100%)
Chloramphenicol
H O
H
(3,4 gl-1 )
OH
H
H OH
HO
HO
Cl
H
O2 N
ß-D-Galactose
Mandel
40-50°C, 24-72h,
(RH) (TG)
Cl
N
H
Amidalkohol (24 gl-1)
-1
(Mandel RH 62 gl )
Si
Si
2-(Trimethylsilyl)-
sek.-Silylalkohol
-1
ethanol (22,3 gl )
(Mandel RH 3,0 gl-1 )
(0,7 gl-1 )
I!L!
Biotechnology!
Synthesis of GOS in sweet whey
-  conditions: whey concentrate, 37°C
-  industrial reference enzyme: Bacillus circulans
Content of lactose and GOS [g/L]
180
lactose
160
Content of lactose and GOS [g/L]
B. circulans* β-galactosidase
200
GOS
140
120
100
80
60
40
20
Metagenome-M1-β-galactosidase
200
180
lactose
160
GOS
140
120
100
80
60
40
20
0
0
0
5
10
15
time [h]
20
25
30
0
5
10
15
20
25
time [h]
* US Patent 4,237,230 Iida et al.
new metagenome enzyme M1 is >40% better in yield
©Prof.
Dr. Lutz Fischer
30
I!L!
Biotechnology! Carbohydrate Epimerases (EC 5, Isomerases)
q  >20 types of carbohydrate
epimerases known*
q  reaction on C1, C2, C3, C4, C5
or C6 of sugar moieties
q  investigated in Pharmaindustry
for drug-design
q  cellobiose 2-epimerases
-  only found in bacteria
-  Ruminococus albus
substrates: cellobiose,
lactose, β-mannose,
globotriose
*Ito (2010) J. Appl. Glycosci. 57, 1-6
Dr. Lutz Fischer
©Prof.
I!L!
Biotechnology!
Cellobiose 2-Epimerase (EC 5.1.3.11)
q  epilactose (4-O-β-Gal-Man):
-  contained in chemically synthesized lactulose preparations (pharma)
-  production by cellobiose 2-epimerase from Ruminococus albus
(pHoptimum 7,5 - 8,0)
Lactose
(4-O-D-Gal-D-Glu)
40
60
Epilactose
(4-O-D-Gal-D-Man)
-  intervention tests with rats*:
à health-promoting properties, stimulation of bifidobacteria growth,
fascilation of mineral absorption and more
-  prebiotic properties*
©Prof.
Dr. Lutz Fischer
*Watanabe et al. (2008) J. Dairy Sci. 91(12), 4518-25
I!L!
Biotechnology!
Cellobiose 2-epimerase: Lactulose* (???)
Lactose
(4-O-β-D-Gal-D-Glu)
?
?
Epilactose
(4-O-β-D-Gal-D-Man)
+
Lactulose
(4-O-β-D-Gal-D-Fru)
q  thermostable cellobiose 2-epimerase from Caldicellulosiruptor
saccharolyticus recombinant in E. coli (pET vector), 16°C, 16h,
shaking flask
q  synthesis of epilactose and lactulose in 200 µL scale (!!!), pH 7.5,
700 gL-1 lactose, at 80°C over 2h
q  inacceptable scientific offence: products ONLY determined by
HPLC/RI detector à structure not proven, other stereoisomers ???
©Prof.
Dr. Lutz Fischer
*Kim and Oh (2012) Bioresource Technol. 104, 668-72
I!L!
Biotechnology! Cellobiose 2-Epimerase: C. saccharolyticus*
lactulose
epilactose
©Prof.
Dr. Lutz Fischer
*Kim and Oh (2012) Bioresource Technol. 104, 668-72
I!L!
Biotechnology! Cellobiose 2-Epimerase: C. saccharolyticus*
Are these results really true/likely ... ?
The enzyme was patented by Watanabe et al. (2010), Japan
©Prof.
Dr. Lutz Fischer
*Kim and Oh (2012) Bioresource Technol. 104, 668-72
I!L!
Biotechnology!
Marktgerechte NEUE Enzyme ???
q  sollen zukünftig NEUE rekombinante Enzyme für die
Lebensmittelindustrie zur Verfügung stehen ?
à Weiterentwicklung von „Food-Grade“ Expressionssystemen
q  sollen zukünftig NEUE BIO-Enzyme für die
Lebensmittelindustrie zur Verfügung stehen ?
-  Ressource: essbare pflanzliche (tierische?) Rohwaren
-  Screening nach endogenen,
anwendungsrelevanten Enzymen
in Lebensmittelrohwaren,
die qualitativ und quantitativ
zur Verfügung stehen
©Prof.
Dr. Lutz Fischer
I!L!
Benefits of Recombinant Enzyme Production
Biotechnology!
q  controlled manufacturing plants under genetic engineering act
(à ecology)
q  production strains and vectors: „food-grade“
(à food safety)
Food
q  microbial „over-expression“ due to known, effective expression systems
(à economy)
q  secretion of enzymes into the medium
(à economy, quality)
q  enzyme preparations with less/no side-activities
(à quality, sensoric aspects, shelf life of products)
q  novel enzymes with desired properties
(à quality, innovations)
©Prof.
Dr. Lutz Fischer
I!L!
Biotechnology!
Acknowledgment
BMBF
BioChance PLUS-3
FKZ 0313137
BRAIN AG
©Prof.
Dr. Lutz Fischer
AiF-FV 16541 N
AiF-FV 15801 N
AiF-FV 14787 N

785 - DGMK

Colilert*-18 - Idexx

October 2014 Deadline, Key Actions 1,2,3

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