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

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

How do psychrophiles maintain appropriate rate of reactions ?

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

Cells and extracellular ice

Bacteria in liquid veins of sea ice

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

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.

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

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

Animation of the process of ice recrystallization

Some cryoprotectants Glucose Tréhalose Sorbitol Ribitol Glycérol

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

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

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

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

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

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

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

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+ 3-D-galactonolactone + NADH + H+   -galactosidase [Lactose ] Free[D-galactose] [Lactose] hydrolysed (mg/l) (mg/l) (mg/l) P. haloplanktis 0.778 0.135 0.256 Maxilact 0.770 0.049 0.093

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

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

Cold 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)

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)