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)