psychrophiles: a challenge for life laboratory of biochemistry belgium laboratory of biochemistry...
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Psychrophiles: a challenge for lifePsychrophiles: a challenge for life
Laboratory of BiochemistryBelgiumLaboratory of BiochemistryBelgium Punta del Este, May 2010Punta del Este, May 2010
Arrhenius law: Thermodependence of reaction rateArrhenius law: Thermodependence of reaction rate
k=A. exp (–Ea/RT)
10 20 30 40 50 T(°C)
k
Q10=VT+10/VT=2-3
How do psychrophiles maintainHow do psychrophiles maintain appropriate rate of reactionsappropriate rate of reactions ??
How do psychrophiles, living belowHow do psychrophiles, living below 0°C, prevent freezing?0°C, prevent freezing?
Cells and extracellular ice
Bacteria in liquid veins of sea ice
General properties of enzymes from psychrophiles
General properties of enzymes from psychrophiles
• High specific activity at low and moderate temperatures
• High thermosensitivity - Flexible structure
• High specific activity at low and moderate temperatures
• High thermosensitivity - Flexible structure
AHAPPA
Specific activities of psychrophilic(blue) and mesophilic(green) enzymes as a function of temperature
Specific activities of psychrophilic(blue) and mesophilic(green) enzymes as a function of temperature
Growth of an Antarctic bacterium (left) and excretion of Growth of an Antarctic bacterium (left) and excretion of α- α- amylase amylase (right) as a function of temperature(right) as a function of temperature
4°C18°C
25°C
4°C
18°C
25°C25°C
Growth α-α-α-amylase
β-galactosyl-1, 3,-α-N-acetyl glucosamine
Antifreeze GlycoProtein(AFGP)
Animation of the process of ice recrystallization
Some cryoprotectants
Glucose Tréhalose
Glycérol Sorbitol Ribitol
Effects of cryoprotectantsEffects of cryoprotectants
1- They depress the freezing point of water
2-They prevent cell dehydration when extracellular ice is present
3-They protect proteins against cold denaturation
Water molecules
Homogeneous ice nucleation
(-39°C)
Heterogeneous ice nucleation
(>-5°C)
INP ANP
Stimulation Inhibition
Ice crystals O
AFP (IR)
Growth inhibition and recrystallization inhibition
Effects of ice-nucleating proteins
1-Ice is confined in the extracellular space
2-The formation of ice is controlled by the organism (time)
3-At the same time production of cryoprotectants
4-Some water is lost contributing to decrease the freezing point in the intracellular space
Water molecules
Homogeneous ice nucleation
(-39°C)
Heterogeneous ice nucleation
(>-5°C)INP ANP
Stimulation Inhibition
Ice crystals O
AFP (IR)
Growth inhibition and recrystallization inhibition
Biodegradation of polluted water by Biodegradation of polluted water by Arthrobacter psychrolactophilus Arthrobacter psychrolactophilus at 10°Cat 10°C
Open symbols: sterilized
Black symbols: with strain
a: turbidity
b: protein concentration
c: amino acids
d: simple sugars
Turbidity
Proteins
Amino acids
Simple sugars
Psychrophiles and cold-active enzymes in BiotechnologyPsychrophiles and cold-active enzymes in BiotechnologyApplications Advantage Involved enzymesDetergents Washing at low temperature Protease, lipase, amylase,
cellulase, oxygenase
Food industry Improved taste and aroma of fermentation products (cheese,etc)
Fermentation and ripening enzymes
Lactose hydrolysis Β-galactosidase
Juice clarification and yield of extraction
Pectinase, cellulase
Meat tenderization Protease
Organic synthesis Volatile and heat-sensitive compounds, organic phase synthesis
Protease, lipase, glycosyl- hydrolase, etc
Molecular biology Mild inactivation, ligation, PCR Various enzymes
Low temperature Ligation, carry-over in PCR
Textiles Improved quality due to mild heat treatment for desizing, bio-polishing and stone- washing of fabrics
Amylase, laccase, cellulase
Environment In situ bioremediation of organic pollutants and hydrocarbons
Various hydrolases, mono- and-dioxygenases, dehydrogenases
Biogas production Psychrophilic anaerobic digestion
Lactose splitting catalysed by beta-galactosidase (lactase)
lactase+Lactase
Lactozym(Novo), Maxilact(Gist Brocades)
Tests in milk at 5 °CTests in milk at 5 °C
ß-galactosidaseß-galactosidase
Lactose + HLactose + H22O O D-glucose + D-galactoseD-glucose + D-galactose
Gal-DHGal-DHD-galactose + NADD-galactose + NAD+ + 3-D-galactonolactone + NADH + H3-D-galactonolactone + NADH + H++
ß-galactosidaseß-galactosidase
Lactose + HLactose + H22O O D-glucose + D-galactoseD-glucose + D-galactose
Gal-DHGal-DHD-galactose + NADD-galactose + NAD+ + 3-D-galactonolactone + NADH + H3-D-galactonolactone + NADH + H++
-galactosidase-galactosidase [Lactose ] [Lactose ] Free[D-galactose]Free[D-galactose] [Lactose] hydrolysed [Lactose] hydrolysed (mg/l) (mg/l) (mg/l) (mg/l) (mg/l)(mg/l)
P. haloplanktisP. haloplanktis 0.7780.778 0.135 0.135 0.2560.256
MaxilactMaxilact 0.770 0.770 0.049 0.049 0.093 0.093
-galactosidase-galactosidase [Lactose ] [Lactose ] Free[D-galactose]Free[D-galactose] [Lactose] hydrolysed [Lactose] hydrolysed (mg/l) (mg/l) (mg/l) (mg/l) (mg/l)(mg/l)
P. haloplanktisP. haloplanktis 0.7780.778 0.135 0.135 0.2560.256
MaxilactMaxilact 0.770 0.770 0.049 0.049 0.093 0.093
Isomerisation of D-galactose into D-tagatose (sweetener)Isomerisation of D-galactose into D-tagatose (sweetener)
D-galactose D-tagatose
Ca(OH)2
3D – Structure of cold-adapted xylanase3D – Structure of cold-adapted xylanase
Family 11 XylanaseFamily 10 Xylanase Family 8 Endoglucanase CelA
Cold xylanase in bakingCold xylanase in baking
Xylanase Xyl/Kg Loaf Vol Cut Width
(IU) (ml) (mm)
1 Control 0.00 1675 19
2 Belase 31.50 1900 27
3 pXyl 0.25 1900 34
4 pXyl 0.50 1925 30
J-S Dumont d’Urville (Terre Adélie)
AcknowledgmentsAcknowledgmentsAcademic: Richard Haser (Lyon):X-ray crystallography
Nick Russell (London): Microbiology, Biochemistry
Luisa Tutino (Naples): Expression in psychrophiles
Rosa Margesin (Innsbruck): Biotechnology
Rick Cavicchioli (Sydney): Microbiology, Biochemistry
Jozef Van Beeumen (Gent): Biochemistry, Crystallography
Philip Thonnart (Gembloux): Biotechnology
Industrial: Beldem-Puratos (Belgium): Thierry Dauvrin and Jacques Georis
Nutrilab (Belgium):
In the Antarctic ( J-S Dumont d’Urville and Kerguelen): Expeditions polaires francaises
In the Arctic (Ny-Alesund-Dirigible Italia): CNR (Italy)
Academic: Richard Haser (Lyon):X-ray crystallography
Nick Russell (London): Microbiology, Biochemistry
Luisa Tutino (Naples): Expression in psychrophiles
Rosa Margesin (Innsbruck): Biotechnology
Rick Cavicchioli (Sydney): Microbiology, Biochemistry
Jozef Van Beeumen (Gent): Biochemistry, Crystallography
Philip Thonnart (Gembloux): Biotechnology
Industrial: Beldem-Puratos (Belgium): Thierry Dauvrin and Jacques Georis
Nutrilab (Belgium):
In the Antarctic ( J-S Dumont d’Urville and Kerguelen): Expeditions polaires francaises
In the Arctic (Ny-Alesund-Dirigible Italia): CNR (Italy)