Cold-adapted alanine dehydrogenases from two antarctic bacterial strains: gene cloning, protein characterization, and comparison with mesophilic and thermophilic counterparts.

The genes encoding NAD(+)-dependent alanine dehydrogenases (AlaDHs) (EC 1.4.1.1) from the Antarctic bacterial organisms Shewanella sp. strain Ac10 (SheAlaDH) and Carnobacterium sp. strain St2 (CarAlaDH) were cloned and expressed in Escherichia coli. Of all of the AlaDHs that have been sequenced, SheAlaDH exhibited the highest level of sequence similarity to the AlaDH from the gram-negative bacterium Vibrio proteolyticus (VprAlaDH). CarAlaDH was most similar to AlaDHs from mesophilic and thermophilic Bacillus strains. SheAlaDH and CarAlaDH had features typical of cold-adapted enzymes; both the optimal temperature for catalytic activity and the temperature limit for retaining thermostability were lower than the values obtained for the mesophilic counterparts. The k(cat)/K(m) value for the SheAlaDH reaction was about three times higher than the k(cat)/K(m) value for VprAlaDH, but it was much lower than the k(cat)/K(m) value for the AlaDH from Bacillus subtilis. Homology-based structural models of various AlaDHs, including the two psychotropic AlaDHs, were constructed. The thermal instability of SheAlaDH and CarAlaDH may result from relatively low numbers of salt bridges in these proteins.     

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.     

The effect of the carbon sources and calcium ions on the activity of thermostable alpha-amylase in Bacillus sp. 86

Amylolytic activity of Bacillus sp. 86 thermophilic strain was studied as affected by carbon sources. Glucose and saccharose being added to the nutrition medium with baker's yeast as the basic source of carbon the activity of alpha-amylase decreased. Introduction of native or soluble starch to the medium promoted a sharp increase in the enzymic activity. Bacillus sp. 86 being periodically cultivated in the optimized medium containing 4% of soluble starch and 0.5% of CaCl2, an eight-fold increase of amylolytic activity of cultural liquid was achieved as compared to the enzyme activity in the primary medium.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost

Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C. At 65 degrees C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.     

Determination of the kinetic parameters of the phenol-degrading thermophile Bacillus themoleovorans sp. A2

Phenolic compounds are pollutants in many wastewaters, e.g. from crude oil refineries, coal gasification plants or olive oil mills. Phenol removal is a key process for the biodegradation of pollutants at high temperatures because even low concentrations of phenol can inhibit microorganisms severely. Bacillus thermoleovorans sp. A2, a recently isolated thermophilic strain (temperature optimum 65 degrees C), was investigated for its capacity to degrade phenol. The experiments revealed that growth rates were about four times higher than those of mesophilic microorganisms such as Pseudomonas putida. Very high specific growth rates of 2.8 h(-1) were measured at phenol concentrations of 15 mg/l, while at phenol concentrations of 100-500 mg/l growth rates were still in the range of 1 h(-1). The growth kinetics of the thermophilic Bacillus thermoleovorans sp. A2 on phenol as sole carbon and energy source can be described using a three-parameter model developed in enzyme kinetics. The yield coefficient Yx/s of 0.8-1 g cell dry weight/g phenol was considerably higher than cell yields of mesophilic bacteria (Yx/s 0.40-0.52 g cell dry weight/g phenol). The highest growth rate was found at pH 6. Bacillus thermoleovorans sp. A2 was found to be insensitive to hydrodynamic shear stress in stirred bioreactor experiments (despite possible membrane damage caused by phenol) and flourished at an ionic strength of the medium of 0.25(-1) mol/l (equivalent to about 15-60 g NaCl/l). These exceptional properties make Bacillus thermoleovorans sp. A2 an excellent candidate for technical applications.     

Thermoalkalophilic lipase-producing Bacillus selected by continuous cultivation

Abstract Several lipase-producing thermophilic bacteria were isolated by continuous culture from samples collected near Bulgarian hot springs. Most of them had lipase activity of about 0.5 U ml⁻¹ when activated in shaken flasks. Three strains, Gram-positive sporeforming rods, possess higher enzyme activity in a Tween-80 containing medium. The highest lipase activity (2.0–3.0 U ml⁻¹) was observed in the newly-isolated thermoalkalophilic Bacillus sp. strain MC7.     

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.     

Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60 degrees C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70 degrees C, respectively, and showed optimal activity around at 70 degrees C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.     

Description of heterotrophic bacteria occurring in paper mills and paper products

AIMS: To isolate aerobic mesophilic bacilli and thermophilic bacteria from different paper mill samples and to evaluate their potential harmfulness. METHODS AND RESULTS: A total of 109 mesophilic and 68 thermophilic isolates were purified and characterized by automated ribotyping and partial 16S rDNA sequencing. The mesophilic isolates belonged to the genera Bacillus (13 taxa), Brevibacillus (three taxa) and Paenibacillus (five taxa). The thermophilic bacteria represented seven taxa of Bacillus, Geobacillus or Paenibacillus, four of proteobacteria and one of actinobacteria. The most frequently occurring bacteria were Bacillus cereus, B. licheniformis, Pseudoxanthomonas taiwanensis and bacteria closely related to Paenibacillus stellifer, P. turicensis or Leptothrix sp. One mill was contaminated throughout with bacteria of a novel mesophilic genus most closely related to Brevibacillus centrosporus and another with bacteria of a novel thermophilic genus most closely related to Hydrogenophilus thermoluteolus. One B. cereus isolate producing haemolytic diarrhoeal enterotoxin was detected and all the tested B. licheniformis isolates produced a metabolite toxic to boar sperm cells. CONCLUSIONS: The bacilli and thermophilic bacteria isolated represent species which should not present occupational hazards in paper mill environments. The most harmful bacterium detected was B. licheniformis and potentially also B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the microbial diversity in a paper mill provides a rational basis for development of an effective controlling programme. A database constructed from the fingerprints generated using automated ribotyping helps to identify and trace the contamination routes of bacteria occurring in paper mills.     

Suitability of different β-galactosidases as reporter enzymes in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The suitability of three β-galactosidases as reporter enzymes for promoter expression analyses was investigated in Bacillus subtilis with respect to various temperature conditions during cultivation and assay procedures. Starting from the hypothesis that proteins derived from diverse habitats have different advantages as reporters at different growth temperatures, the beta-galactosidases from the thermophilic organism Bacillus stearothermophilus, from the mesophilic bacterium Escherichia coli and from the psychrophilic organism Pseudoalteromonas haloplanktis TAE79 were analysed under control of the constitutive B. subtilis lepA promoter. Subsequent expression of the β-galactosidase genes and determination of specific activities was performed at different cultivation and assay temperatures using B. subtilis as host. Surprisingly, the obtained results demonstrated that the highest activities over a broad cultivation temperature range were obtained using the β-galactosidase from the mesophilic bacterium E. coli whereas the enzymes from the thermophilic and psychrophilic bacteria revealed a more restricted usability in terms of cultivation temperature.     

Toxicity of paraquat to microorganisms.

The biochemical response of the microorganisms Lipomyces starkeyi (Lod & Rij), Escherichia coli K-12 W3110, Bacillus subtilis 168 (Marburg) and Pseudomonas sp. strain TTO1 to the presence of growth-inhibitory concentrations of paraquat was studied. Paraquat was added to each culture at a concentration previously determined to reduce the culture growth rate by up to 50%. The changes in activity of a number of enzymes previously shown to be associated with the defense of the mammalian system against the action of paraquat were studied. While the response of E. coli was in agreement with that found in other studies of this microorganism and supports a commonly accepted mechanism for paraquat toxicity, the results obtained with L. starkeyi, B. subtilis, and Pseudomonas sp. strain TTO1 suggest that other mechanisms exist for protection against the toxicity of paraquat.     

Toxicity of paraquat to microorganisms

The biochemical response of the microorganisms Lipomyces starkeyi (Lod & Rij), Escherichia coli K-12 W3110, Bacillus subtilis 168 (Marburg) and Pseudomonas sp. strain TTO1 to the presence of growth-inhibitory concentrations of paraquat was studied. Paraquat was added to each culture at a concentration previously determined to reduce the culture growth rate by up to 50%. The changes in activity of a number of enzymes previously shown to be associated with the defense of the mammalian system against the action of paraquat were studied. While the response of E. coli was in agreement with that found in other studies of this microorganism and supports a commonly accepted mechanism for paraquat toxicity, the results obtained with L. starkeyi, B. subtilis, and Pseudomonas sp. strain TTO1 suggest that other mechanisms exist for protection against the toxicity of paraquat.     

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.     

Molecular and biochemical characterization of a novel intracellular low-temperature-active xylanase

A 990 bp full-length gene (xynAHJ2) encoding a 329- residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperatureactive enzyme (exhibiting optimum activity at 35 degrees C, 62% at 20 degrees C, and 38% at 10 degrees C; thermolability at > or =45 degrees C). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to Ni2+, Ca2+, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperatureactive xylanase.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

Production and properties of a bacterial thermostable exo-inulinase

Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11 strain was studied by batch cultivation in a fermentor. The achieved inulinase and invertase activities after a short growth time (4.25 h) were similar or higher compared to those reported for other mesophilic aerobic or anaerobic thermophilic bacterial producers and yeasts. The investigated enzyme belonged to the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could be used at temperatures above 65 degrees C and pH range 5.5-7.5. The obtained crude enzyme preparation possessed high thermostability. The residual inulinase and invertase activities were 92-98% after pretreatment at 65 degrees C for 60 min in the presence of substrate inulin.