Pressure assisted stabilization of biocatalysts at elevated temperatures: Characterization by dynamic light scattering

The effect of pressure, at elevated temperatures, is reported on the activity and stability of a thermophilic endo‐β‐glucanase from the filamentous fungus Talaromyces emersonii. The production of reduced sugars after treatment at different temperatures and pressures is used as a measure of the activity and stability of the enzyme. The activity of the enzyme is maintained to higher temperatures with increasing pressure. For example, the relative activity of endo‐β‐glucanase decreases to 30% after 4 h at 75°C and 1 bar, whereas it is preserved at 100% after 6 h at 75°C and 230 bar. High‐pressure dynamic light scattering is used to characterize the hydrodynamic radius of the enzyme as a function of pressure, temperature, and time. At higher temperature the hydrodynamic radius increases with time, whereas increasing pressure suppresses this effect. Changes in the hydrodynamic radius are correlated with the activity measurements obtained at elevated pressures, since the changes in the hydrodynamic radius indicate structural changes of the enzyme, which cause the deactivation. Biotechnol. Bioeng. 2013; 110: 1674–1680. © 2012 Wiley Periodicals, Inc.

[1]  B. Adney,et al.  Measurement of Cellulase Activities; LAP-006 NREL Analytical Procedure , 1996 .

[2]  E. Mombelli,et al.  Exploring hyperthermophilic proteins under pressure: theoretical aspects and experimental findings. , 2002, Biochimica et biophysica acta.

[3]  J. Reyes-De-Corcuera,et al.  High pressure enhancement of enzymes: A review , 2009 .

[4]  U. Kulozik,et al.  Structure and stabilizing interactions of casein micelles probed by high-pressure light scattering and FTIR. , 2011, The journal of physical chemistry. B.

[5]  D. Clark,et al.  Pressure effects on activity and stability of hyperthermophilic enzymes. , 2001, Methods in enzymology.

[6]  S. Provencher A constrained regularization method for inverting data represented by linear algebraic or integral equations , 1982 .

[7]  M. Ribeiro,et al.  Pressure-enhanced activity and stability of α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase , 2010 .

[8]  P. Masson,et al.  High pressure effects on protein structure and function , 1996, Proteins.

[9]  D. Clark,et al.  Pressure Stabilization of Proteins from Extreme Thermophiles , 1994, Applied and environmental microbiology.

[10]  D. Combes,et al.  Increased thermostability of three mesophilic β-galactosidases under high pressure , 1997, Biotechnology Letters.

[11]  Rainer Jaenicke,et al.  Proteins under pressure , 1994 .

[12]  V. V. Mozhaev,et al.  [Inactivation and reactivation of proteins (enzymes)]. , 1982, Molekuliarnaia biologiia.

[13]  D. Millar,et al.  High pressure stabilization of acetylcholinesterase sizeozymes against thermal denaturation. , 1974, Biophysical chemistry.

[14]  Shangtian Yang,et al.  A Dynamic Light Scattering Study of β‐Galactosidase: Environmental Effects on Protein Conformation and Enzyme Activity , 1994 .

[15]  S. Provencher CONTIN: A general purpose constrained regularization program for inverting noisy linear algebraic and integral equations , 1984 .

[16]  P. Masson,et al.  Exploiting the effects of high hydrostatic pressure in biotechnological applications , 1994 .

[17]  I. Park,et al.  High-pressure dynamic light scattering of Poly(ethylene-co-1-butene) in ethane, propane, butane, and pentane at 130°C and kilobar pressures , 2004 .

[18]  J. Sengers,et al.  Dynamics of critical fluctuations in polymer solutions. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[19]  D. Clark,et al.  Pressure effects on intra- and intermolecular interactions within proteins. , 2002, Biochimica et biophysica acta.

[20]  R. Buckow,et al.  Effect of high hydrostatic pressure-temperature combinations on the activity of β-glucanase from barley malt , 2005 .

[21]  N. Smolin,et al.  Thermal breaking of spanning water networks in the hydration shell of proteins. , 2005, The Journal of chemical physics.

[22]  C. Zetzl,et al.  Development of an integrated thermal and enzymatic hydrolysis for lignocellulosic biomass in fixed-bed reactors , 2011 .

[23]  R. Anantheswaran,et al.  Effect of high-pressure processing on activity and structure of alkaline phosphatase and lactate dehydrogenase in buffer and milk. , 2007, Journal of agricultural and food chemistry.

[24]  Jerson L. Silva,et al.  Pressure stability of proteins. , 1993, Annual review of physical chemistry.

[25]  D. Clark,et al.  Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii , 1995, Applied and environmental microbiology.

[26]  R. Winter,et al.  Exploring the temperature–pressure configurational landscape of biomolecules: from lipid membranes to proteins , 2005, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences.

[27]  M. Gross,et al.  Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes. , 1994, European journal of biochemistry.