1. Psychrophiles: a challenge for life
Laboratory of Biochemistry
Belgium
Punta del Este, May 2010

2. Arrhenius law: Thermodependence of reaction rate
k=A. exp (–Ea/RT) k
Q10=VT+10/VT=2-3
T(°C) 10 20 30 40 50

3. How do psychrophiles maintain appropriate rate of reactions ?

4. How do psychrophiles, living below 0°C, prevent freezing?

5. Cells and extracellular ice

6. Bacteria in liquid veins of sea ice

8. General properties of enzymes from psychrophiles
High specific activity at low and moderate temperatures
High thermosensitivity - Flexible structure

9. Specific activities of psychrophilic(blue) and mesophilic(green) enzymes as a function of temperature
PPA
AHA
On this plot, you can see the effect of temperature on the activity of two homologous enzymes: AHA is the alpha-amylase from Pseudoalteromonas haloplanktis, an antarctic bacteria; and PPA is the alpha-amylase from the pig pancreas. This graph reveals 3 basic features:
1. Psychrophiles synthesise enzymes with higher specific activity at low temperatures.
2. The apparent maximal activity is shifted toward low temperatures, reflecting their weak thermostability.
3. The adaptation is apparently not complete. The specific activity displayed by the psychrophilic enzymes around 0°C remains lower than that of the mesophilic at 37°C.

10. Growth of an Antarctic bacterium (left) and excretion of α-
Growth of an Antarctic bacterium (left) and excretion of α amylase (right) as a function of temperature
α-amylase
Growth α- α-
4°C
4°C
18°C
25°C
18°C
25°C
25°C

11. Antifreeze GlycoProtein(AFGP)
β-galactosyl-1, 3,-α-N-acetyl glucosamine

13. Animation of the process of ice recrystallization

14. Some cryoprotectants
Glucose
Tréhalose
Sorbitol
Ribitol
Glycérol

15. Effects 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

16. Homogeneous ice nucleation Heterogeneous ice nucleation
Water molecules
Homogeneous ice nucleation
(-39°C)
Heterogeneous ice nucleation
(>-5°C)
INP
ANP
Stimulation
Inhibition O
Ice crystals
AFP (IR)
Growth inhibition and recrystallization inhibition

17. 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

18. INP ANP Stimulation Inhibition O Ice crystals AFP (IR) Water molecules
Homogeneous ice nucleation
(-39°C)
Heterogeneous ice nucleation
(>-5°C)
INP
ANP
Stimulation
Inhibition O
Ice crystals
AFP (IR)
Growth inhibition and recrystallization inhibition

19. b: protein concentration
Biodegradation of polluted water by Arthrobacter psychrolactophilus at 10°C
Turbidity
Open symbols: sterilized
Black symbols: with strain
a: turbidity
Proteins
b: protein concentration
c: amino acids
d: simple sugars
Amino acids
Simple sugars

20. Psychrophiles and cold-active enzymes in Biotechnology
Applications
Advantage
Involved enzymes
Detergents
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

21. Lactose splitting catalysed by beta-galactosidase (lactase)
Lactozym(Novo), Maxilact(Gist Brocades)

22. Tests in milk at 5 °C ß-galactosidase
Tests in milk at 5 °C
ß-galactosidase
Lactose + H2O D-glucose + D-galactose
  Gal-DH
D-galactose + NAD D-galactonolactone + NADH + H+
-galactosidase [Lactose ] Free[D-galactose] [Lactose] hydrolysed
(mg/l) (mg/l) (mg/l)
P. haloplanktis
Maxilact

23. Isomerisation of D-galactose into D-tagatose (sweetener)
Ca(OH)2
D-galactose
D-tagatose

24. 3D – Structure of cold-adapted xylanase
The structure determined is shown here and can be described as a distorted (α/α)6 barrel, that is, it consists of six inner helices and six outer helices in addition to a number of beta strands. The active site is located in a long acidic cleft running across the molecular surface at the N-terminal end of the inner helices with the the N and C termini being located on the opposite side of the structure to this. Clearly, this structure is very different to that described for the family 10 and 11 xylanases but is similar to that for the family 8 endoglucanase CelA.
Family 8 Endoglucanase CelA
Family 11 Xylanase
Family 10 Xylanase

25. Cold xylanase in baking
Xylanase Xyl/Kg Loaf Vol Cut Width
(IU) (ml) (mm)
1 Control
2 Belase
3 pXyl
4 pXyl

26. J-S Dumont d’Urville (Terre Adélie)

27. Acknowledgments 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)