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

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Presentation on theme: "Psychrophiles: a challenge for life Laboratory of Biochemistry Belgium Laboratory of Biochemistry Belgium Punta del Este, May 2010."— Presentation transcript:

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

2 Arrhenius law: Thermodependence of reaction rate Arrhenius law: Thermodependence of reaction rate k=A. exp (–Ea/RT) T(°C) k Q 10 =V T+10 /V T =2-3

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

7

8 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

9 AHA PPA Specific activities of psychrophilic(blue) and mesophilic(green) enzymes as a function of temperature

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

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

12

13 Animation of the process of ice recrystallization

14 Some cryoprotectants Glucose Tréhalose Glycérol SorbitolRibitol

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 Water molecules Homogeneous ice nucleation (-39°C) Heterogeneous ice nucleation (>-5°C) INPANP StimulationInhibition Ice crystals O 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 Water molecules Homogeneous ice nucleation (-39°C) Heterogeneous ice nucleation (>-5°C) INPANP StimulationInhibition Ice crystals O AFP (IR) Growth inhibition and recrystallization inhibition

19 Biodegradation of polluted water by Arthrobacter psychrolactophilus at 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

20 Psychrophiles and cold-active enzymes in Biotechnology ApplicationsAdvantageInvolved 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, PCRVarious enzymes Low temperatureLigation, 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 productionPsychrophilic anaerobic digestion

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

22 Tests in milk at 5 °C ß-galactosidase Lactose + H 2 O D-glucose + D-galactose Gal-DH Gal-DH D-galactose + NAD + 3-D-galactonolactone + NADH + H + ß-galactosidase Lactose + H 2 O D-glucose + D-galactose Gal-DH Gal-DH D-galactose + NAD + 3-D-galactonolactone + NADH + H + -galactosidase [Lactose ]Free[D-galactose] [Lactose] hydrolysed -galactosidase [Lactose ]Free[D-galactose] [Lactose] hydrolysed (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) P. haloplanktis Maxilact galactosidase [Lactose ]Free[D-galactose] [Lactose] hydrolysed -galactosidase [Lactose ]Free[D-galactose] [Lactose] hydrolysed (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) P. haloplanktis Maxilact

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

24 3D – Structure of cold-adapted xylanase Family 11 XylanaseFamily 10 Xylanase Family 8 Endoglucanase CelA

25 Cold xylanase in baking Xylanase Xyl/KgLoaf VolCut Width (IU) (ml) (mm) 1Control Belase pXyl pXyl

26 J-S Dumont dUrville (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 dUrville 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 dUrville and Kerguelen): Expeditions polaires francaises In the Arctic (Ny-Alesund-Dirigible Italia): CNR (Italy)


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