Thermodynamic theory explains the temperature optima of soil microbial processes and high Q10 values at low temperatures

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory – MMRT (Hobbs et al., 2013 , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q₁₀ calculations) at low temperatures and that Q₁₀ declined as temperature increases in agreement with data synthesis from the literature. Declining Q₁₀ and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme‐catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems – a fundamental component of models for climate change.     

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.     

The isomaltulose synthesising enzyme ofSerratia plymuthica

Summary An intracellular enzyme was located inSerratia plymuthica which produced isomaltulose from sucrose. The enzyme was purified giving a preparation with a specific activity of 1,285. It has pH and temperature optima of 6.0 and 30°C, respectively. The enzyme was stable retaining 100% activity after 2 weeks at 30°C. It had an isoelectric point at pH 9.0, a Mr of 79,500 and the Km for sucrose was 65.3mM. The enzyme converted 40% (w/v) sucrose to isomaltulose with an efficiency of 87%.     

Temperature and pH optima of enzyme activities produced by cellulolytic thermophilic fungi in batch and solid-state cultures

Summary The temperature and pH optima of cellulolytic activities produced by thermophilic fungi in liquid and solid-state cultures were established. Some differences in optimal conditions for enzyme activities, which depended on culture methods, were confirmed.     

Chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima

A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg(2+) for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a K(m) of 11.7 microM, a turnover k(cat) of 6.8 s(-1), and a catalytic efficiency of k(cat)/K(m) = 5.8 x 10(5) M(-1) s(-1). dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37 degrees C) than PBCV-1 dUTPase (50 degrees C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81-->Ser81 and Thr84-->Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84-->Arg84, Glu81-->Ser81, and Glu81-->Ser81 plus Thr84-->Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55 degrees C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.     

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.     

Antifungal properties of haem peroxidase from Acorus calamus

BACKGROUND AND AIMS: Plants have evolved a number of inducible defence mechanisms against pathogen attack, including synthesis of pathogenesis-related proteins. The aim of the study was to purify and characterize antifungal protein from leaves of Acorus calamus. METHODS: Leaf proteins from A. calamus were fractionated by cation exchange chromatography and gel filtration and the fraction inhibiting the hyphal extension of phytopathogens was characterized. The temperature stability and pH optima of the protein were determined and its presence was localized in the leaf tissues. KEY RESULTS: The purified protein was identified as a class III haem peroxidase with a molecular weight of approx. 32 kDa and pI of 7.93. The temperature stability of the enzyme was observed from 5 degrees C to 60 degrees C with a temperature optimum of 36 degrees C. Maximum enzyme activity was registered at pH 5.5. The pH and temperature optima were corroborated with the antifungal activity of the enzyme. The enzyme was localized in the leaf epidermal cells and lumen tissues of xylem, characteristic of class III peroxidases. The toxic nature of the enzyme which inhibited hyphal growth was demonstrated against phytopathogens such as Macrophomina phaseolina, Fusarium moniliforme and Trichosporium vesiculosum. Microscopic observations revealed distortion in the hyphal structure with stunted growth, increased volume and extensive hyphal branching. CONCLUSIONS: This study indicates that peroxidases may have a role to play in host defence by inhibiting the hyphal extension of invading pathogens.     

The filamentous fungus Aspergillus nidulans produces an α-L-rhamnosidase of potential oenological interest

The production of α- l -rhamnosidase by Aspergillus nidulans has been investigated. In the presence of rhamnose as sole carbon source, this fungus produces an α- l -rhamnosidase of molecular weight 90 kDa. Production of this enzyme is under carbon catabolite repression, apparently by a CreA-independent system. At acidic ambient pH there is an increase in the synthesis of the enzyme which is not related to PacC. Using ρ-nitrophenyl-α- l -rhamnopyranoside as substrate, the enzyme activity in culture filtrates shows pH and temperature optima of 4·5–8 and 40–50 °C, respectively. At the concentrations found in must or wine, enzyme activity was only slightly affected by glucose and SO₂ and partly inhibited by ethanol, indicating a potential for use in wine aroma release.     

Purification of a highly active protease from aMicrosporum species

A method is described for obtaining a highly active proteolytic enzyme from aMicrosporum species. This protease was purified (200-fold) from a cell-free culture medium by concentration with Carbowax, ammonium sulfate fractionation, charcoal and Celite filtration, calcium phosphate gel treatment, and column chromatography. The pH and temperature optima are 6.8 and 35 C respectively. Requirement of one or more free sulfhydryl group(s) for enzyme activity was indicated by inhibition withp-chloromercuric benzoate. Ethylenediaminetetraacetic acid also caused inhibition of proteolytic activity, which suggests involvement of a metal ion. The enzyme appears to be most active in the reduced form;l-cysteine and 2,3 dimercapto-l-propanol doubled the rate of activity. It has an approximate molecular weight of 51,000 to 69,000. The enzyme was highly active on all proteins examined.     

Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus.

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.     

Purification and sequence analysis of a novel NADP(H)-dependent type III alcohol dehydrogenase from Thermococcus strain AN1.

An NADP(H)-dependent alcohol dehydrogenase was isolated from the hyperthermophilic archaeon Thermococcus strain AN1. This enzyme is a homotetramer with a subunit molecular weight of 46,700. The enzyme oxidizes a series of primary linear alcohols but not methanol. The pH and temperature optima with ethanol as the substrate are 6.8 to 7.0 and 85 degrees C, respectively. The enzyme readily reduced acetaldehyde with NADPH as the cofactor. The gene encoding this enzyme has been cloned and sequenced. An open reading frame of 1,218 bp, starting with ATG and ending with TGA, was identified and corresponded to 406 amino acids. Sequence comparisons show that this Thermococcus strain AN1 enzyme has significant homologies with enzymes from the newly defined type III alcohol dehydrogenase family. Thermococcus strain AN1 alcohol dehydrogenase is the first archaeal enzyme belonging to this family.     

Production of dextranase byLipomyces starkeyi

Summary The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.     

Studies on the characterization of the sodium-potassium transport adenosinetriphosphatase. XI. Comparison of kinetic properties of the purified with the impure membrane-bound enzyme from Squalus acanthias

A variety of kinetic parameters have been compared in the membrane-bound and purified forms of the (sodium + potassium)-activated adenosinetriphosphatase (NaK ATPase) from the rectal gland of the spiny dogfish, Squalus acanthias. The kinetic parameters which have been studied have been temperature optima, pH optima, Mg-activation curves, optimum ATP/Mg ratios, K_m for ATP, ouabain-inhibition curves, and Na and K-activation curves. All kinetic parameters were remarkably similar for both forms of the enzyme. This encourages us to believe that information obtained from the pure enzyme can be extrapolated to the enzyme in its native membrane environment and should throw light on the molecular mechanism of Na and K transport.     

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Plastid Protease Activity and Prolamellar Body Transformation during Greening

Two proteases active in and specific to oat etioplasts and up to 24-hour etiochloroplasts, only very slightly contaminated by other cellular compartments are described. The enzyme showed pH optima of 4.2 (acid) and 6.8 (neutral), temperature optima of 50 C and the highest level of enzyme activity was with prolamellar bodies (PLBs) as substrate. Both enzymes showed evidence of a sulfhydryl reagent requirement, particularly for the neutral enzyme. Levels of both proteases increased up to 4 hours of illumination of leaves, and then sharply decreased with the largest differences exhibited by the neutral protease. The pH values in the plastid stroma indicated that the neutral enzyme was likely to be the most important in PLB transformation. A comparison between plastid-associated and extra-plastidic protease activities showed similar properties, except the affinity toward PLBs, which was much higher for plastid proteases (K_m: 0.2 and 1.1 milligrams protein per milliliter, respectively).     

Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.     

Thermostable dextranases: screening, detection and preliminary characterization

Screening methods have been developed for detection of micro-organisms producing thermostable dextranases. They utilize the incorporation of Blue Dextran into agar or liquid culture media for isolation of active dextranase producers growing at temperatures above 55°C. A variety of high temperature environments in sugar factories and naturally occurring thermal water samples were excellent sources of dextranase producers. A number of aerobic and anaerobic thermophilic bacteria, isolated from these sources, were found to produce thermostable dextranases. Dextranases with the greatest thermostability were found in cultures of anaerobic bacteria grown above 65°C. Temperature optima were determined for several crude enzyme preparations, four of which exhibited temperature optima in the range 65–85°C.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Assay of hypoxanthine-guanine phosphoribosyltransferase in human fibroblast lysates: inactivation of nucleotidase.

Assays of the hypoxanthine-guanine phosphoribosyltransferase enzyme (HGPRT: EC 2.4.2.8) in human fibroblast lysates are affected by the presence of a nucleotidase enzyme which converts the reaction product nucleotide to nucleoside. These enzymes, HGPRT and nucleotidase, have substantially different thermostabilities and pH optima, and the nucleotidase activity can be selectively eliminated. The conditions include preheating the cell lysates at 60°C for 10 min, a temperature at which the HGPRT enzyme is relatively stable, and using HGPRT assays buffered at pH 10. Also, we provide evidence that there is no nucleotidase activity in human lymphoblast lysates. Thus, human lymphoblast and preheated fibroblast lysates can be assayed for HGPRT activity by the DEAE-filter disk method.