The specific activities of mesophilic and thermophilic proteinases

1. Most enzymes from extreme thermophiles do not possess higher specific activities than similar enzymes from mesophiles (measured at their respective growth temperatures). 2. However, using protein substrates, the specific activities of thermophilic proteinases are considerably higher than those of most microbial and eukaryotic proteinases. 3. This property could be attributed to purely kinetic influences on the enzyme, to some specific "design" feature of the proteinase, or to the effects of temperature on the substrate. 4. Comparisons of the rates of hydrolysis of large and small substrates by both mesophilic and thermophilic proteinases suggest that temperature-induced changes in substrate susceptibility are a major factor.     

Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.     

Conservation of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains

Abstract Antibodies against the purified octameric l -leucine dehydrogenase (LeuDH) from the mesophilic Bacillus cereus have been used to screen 16 thermophilic Bacillus strains for LeuDH. 4 of these strains, Bacillus sphaericus 461 and Bacillus sp. 405, 406, and 411, showed a particularly strong cross reaction of the partial identity type when examined by Ouchterlony double diffusion assay, thus indicating that they were immunologically related to the B. cereus enzyme. The LeuDH from the thermophilic strains were very stable and highly active at elevated temperatures, and gave a downward bend at about 55°C in the Arrhenius plot. The pH optimum for l -leucine deamination was around pH 11 for all strains examined.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Discrimination of thermophilic and mesophilic proteins

Background

There is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts.

Results

We have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures.

Conclusions

Carefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.

     

Biotransformation of halophenols by a thermophilic Bacillus sp.

The thermophilic Bacillus sp. A2 transformed various halophenols. 2-Chlorophenol, 2-bromophenol, 3-bromophenol and 2-fluorophenol were transformed under resting cell conditions at 60°C to 3-chlorocatechol, 3-bromocatechol, 4-bromocatechol and 3-fluorocatechol, respectively. The hydroxylation of 3-bromophenol occurred at the proximal and distal position relative to the halogen substituent. In complex medium this strain completely transformed 2-chlorophenol and 2-bromophenol at concentrations up to 1 mM. Concomitantly, an accumulation of oxygen-and temperature sensitive halocatechols was observed. 3-Chlorocatechol possesses a half-life of 11.5 h at 60°C and is therefore readily decomposed during incubation. The hydroxylating system was present in phenolgrown cells but not in glucose-grown cells. The hydroxylase activity could also be induced by 2-chlorophenol. The product, 3-chlorocatechol, is not a substrate for the catechol 2,3-dioxygenase.Abbreviations 2-CP 2-chlorophenol - DCP dichlorophenol - TCP trichlorophenol - tetraCP tetrachlorophenol - MIC minimal inhibitory concentration - CF chloride-free - CFG chloride-free plus glucose - CFGY chloride-free plus glycerol - CFP chloride-free plus phenol - CAM chloramphenicol     

Comparative study of energy-transducing properties of cytoplasmic membranes from mesophilic and thermophilic Bacillus species.

The properties of enzymes involved in energy transduction from a mesophilic (Bacillus subtilis) and a thermophilic (B. stearothermophilus) bacterium were compared. Membrane preparations of the two organisms contained dehydrogenases for NADH, succinate, L-alpha-glycerophosphate, and L-lactate. Maximum NADH and cytochrome c oxidation rates were obtained at the respective growth temperatures of the two bacteria. The enzymes involved in the oxidation reactions in membranes of the thermophilic species were more thermostable than those of the mesophilic species. The apparent microviscosities of the two membrane preparations were studied at different temperatures. At the respective optimal growth temperatures, the apparent microviscosities of the membranes of the two organisms were remarkably similar. The transition from the gel to the liquid-crystalline state occurred at different temperatures in the two species. In the two species, the oxidation of physiological (NADH) and nonphysiological (N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate) electron donors led to generation of a proton motive force which varied strongly with temperature. At increasing temperatures, the efficiency of energy transduction declined because of increasing H+ permeability. At the growth temperature, the efficiency of energy transduction was lower in B. stearothermophilus than in the mesophilic species. Extremely high respiratory activities enabled B. stearothermophilus to maintain a high proton motive force at elevated temperatures. The pH dependence of proton motive force generation appeared to be similar in the two membrane preparations. The highest proton motive forces were generated at low external pH, mainly because of a high pH gradient. At increasing external pH, the proton motive force declined.     

Thermostable amylase production by immobilized thermophilic Bacillus sp.

Agar, agarose and alginate immobilized cells of thermophilic Bacillus sp. WN11 produced 7.0, 6.2, and 10.5 U/ml of thermostable amylase in shake flasks, respectively. Alginate entrapped cells released high level of amylase (10.5 U/ml) than freely suspended cells (8.0 U/ml). Amylase production was stable in five successive batches with productivity of 9-12 U/ml for alginate, 5.5-8.8 U/ml for agar, and 4.8-8.0 for agarose immobilized cells.     

Xylanolytic enzyme activities produced by mesophilic and thermophilic actinomycetes grown on graminaceous xylan and lignocellulose

Mesophilic and thermophilic strains of actinomycetes were grown on media containing graminaceous xylan or lignocellulose. Aliquots of the culture fluids were sampled and assayed for enzyme activities involved in the degradation of hemicellulose. Xylanase, acetyl esterase and α-arabinofuranosidase activities could be detected after different times of incubation; their production was also dependent on the growth medium. The highest levels of xylanase activity were found in cultures of strains of Streptomyces, Actinomadura sp. and Saccharomonospora viridis. Streptomyces cyaneus produced the highest amount of arabinofuranosidase whereas acetyl esterase activities were highest in S. cyaneus, S. viridis and Pseudonocardia thermophila .     

Transformation of synthetic pyrethroid insecticides by a thermophilic Bacillus sp

Employing a mineral salts medium containing Tween 80 as the primary carbon source, a strain of Bacillus stearothermophilus was isolated which was able to hydrolyse selected second and third-generation pyrethroids to non-insecticidal products. Of a range of pyrethroid insecticides the trans-isomer of permethrin was the most readily transformed by this microbial isolate, whilst flumethrin was the least. 3-Phenoxybenzoic acid and the respective halovinyl or haloacid moieties were detected as the major hydrolytic products of the pyrethroids. It is believed that 3-phenoxybenzoic acid was formed from 3-phenoxybenzyl alcohol which was not however detected as an intermediate in these systems. 3-Phenoxybenzoic acid was further transformed to 4-hydroxy-3-phenoxybenzoic acid. A potential metabolic pathway has been described.     

Xylan-hydrolysing enzymes from thermophilic and mesophilic fungi

Screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. The relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of -xylosidase. Several strains of Aspergillus awamori and Aspergillus phoenicis were notable in producing high xylanase and -xylosidase and low protease activities. Of the fungl tested, 13 produced activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, feruloyl and coumaroyl substituents from the backbone of xylan polysaccharides as well as endo-1,4--d-xylanase and -1,4-xylosidase. When the growth medium contained oat spelt xylan as carbon source, higher levels of xylanase, -xylosidase and acetyl xylan esterase were found than in cultures containing meadow fescue grass but the latter were richer in ferulic acid and coumaric acid esterases and 4-O-methylglucuronidase. No single organism or carbon source used was capabie of producing high levels of all the debranching enzymes as well as high levels of enzymes capable of cleaving the glycosidic linkages of the xylan backbone. The best ballnce of enzymes was obtained in cultures of A. awamori IMI 142717 and NRRL 2276 and A. phoenicis IMI 214827. Either of these would be suitable for strain improvement studies.The authors are with The Rowett Research Institute. Bucksburn, Aberdeen, AB2 9SB, UK.T.M. Wood is the corresponding author.     

Density discriminates between thermophilic and mesophilic proteins

Despite an intense interest and a remarkable number of studies on the subject, the relationships between thermostability and (primary, secondary and tertiary) structure of proteins are still not fully understood. Here, comparing the protein density – defined by the ratio between the residue number and protein excluded volume – for a set of thermophilic/mesophilic pairs, we provide evidence that this property is connected to the optimal growth temperature. In particular, our results indicate that thermophilic proteins have – in general – a lower density with respect to the mesophilic counterparts, being such a correlation more pronounced for optimal growth temperature differences greater than 40°C. The effect of the protein thermostability changes on the molecular shape is also presented.     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication