Thermostable d-hydantoinase from thermophilic Bacillus stearothermophilus SD-1: characteristics of purified enzyme

One thousand thermophiles isolated from soils were screened for hydantoinase and its thermostability. One thermophilic bacterium that showed the highest thermostability and activity of hydantoinase was identified to be Bacillus stearothermophilus SD-1 according to morphological and physiological characteristics. The hydantoinase of B. stearothermophilus SD-1 was purified to homogeneity via ammonium sulfate fractionation, anion-exchange chromatography, heat treatment, hydrophobic-interaction chromatography, and preparative gel electrophoresis. The relative molecular mass of the hydantoinase was determined to be 126 kDa by gel-filtration chromatography, and a value of 54 kDa was obtained as a molecular mass of the subunit on analytical sodiumdodecylsulfate/polyacrylamide gel electrophoresis. The hydantoinase was strictly d-specific and metal-dependent. The optimal pH and temperature were about 8.0 and 65°C respectively, and the half-life of the d-hydantoinase was estimated to be 30 min at 80°C, indicating the most thermostable enzyme so far.     

d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4°C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

Enzymatic production of <Emphasis Type="SmallCaps">l</Emphasis>-amino acids from the corresponding <Emphasis Type="SmallCaps">dl</Emphasis>-5-substituted hydantoins by <Emphasis Type="Italic">Bacillus fordii</Emphasis> MH602

Bacillus fordii MH602 was newly screened from soil at 45 °C and exhibited high activities of hydantoinase and carbamoylase, efficiently yielding l-amino acids including phenylalanine, phenylglycine and tryptophan with the bioconversion yield of 60–100% from the corresponding dl-5-substituted hydantoins. Hydantoinase activity was found to be cell-associated and inducible. The optimal inducer was dl-5-methylhydantoin with concentration of 0.014 mol L⁻¹ and added to the fermentation medium in the exponential phase of growth. In the production of optically pure amino acids from dl-5-benylhydantoin, the optimal temperature and pH of this reaction were 45–50 °C and 7.5 respectively. The hydantoinase was non-stereoselective, while carmbamoylase was l-selective. The hydantoinase activity was not subject to substrate inhibition, or product inhibition by ammonia. In addition, The activities of both enzymes from crude extract of the strain were thermostable; the hydantoinase and carbamoylase retained about 90% and 60% activity after 6 h at 50 °C, respectively. Since reaction at higher temperature is advantageous for enhancement of solubility and for racemization of dl-5-substituted hydantoins, the relative paucity of l-selective hydantoinase systems, together with the high level of hydantoinase and carbamoylase activity and unusual substrate selectivity of the strain MH602, suggest that it has significant potential applications.     

Isolation and characterization of a thermophilic bacillus strain,that degrades phenol and cresols as sole carbon source at 70 °C

A phenol-degrading thermophilic bacterium, designated Bacillus sp. A2, was isolated from a water and mud sample from a hot spring in Iceland. The aerobic isolate grew optimally on phenol at 65 °C. At 70 °C, 85% of the optimal growth rate was still observed. No growth was observed at 40 °C and 75 °C. Bacillus sp. A2 is a gram-positive spore-forming rod. According to 16S rDNA analysis Bacillus sp. A2 is closely related to Bacillus stearothermophilus, Bacillus kaustophilus and Bacillus thermoleovorans. Bacillus sp. A2 degraded phenol completely in concentrations up to 5 mM. In addition, all three isomers of cresol were utilized as sole carbon and energy sources. The degradation of phenols proceeds via the meta-cleavage pathway and the enzymes involved in its degradation are constitutively expressed. Received: 13 May 1996 / Received revision: 29 July 1996 / Accepted: 12 August 1996     

Induction of cold shock proteins in Bacillus subtilis

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37°C. The B. subtilis cells were cold shocked at 25°C, 20°C, 15°C, and 10°C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl₂.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Production of hydantoinase fromPseudomonas fluorescens strain DSM 84

Summary Pseudomonas fluorescens strain DSM 84 was selected as a good hydantoinase (dihydropyrimidinase E.C. 3.5.2.2.) producer from a screening involving 60 collection strains. Optimization of the culture and growth conditions were performed in order to increase the enzyme production. A mineral medium supplemented with 10 g/l of yeast extract having an initial pH of 7.1±0.1 but containing no additional carbon source or inducer was devised. The strain DSM 84 was found to produce the maximal level of hydantoinase in the defined mineral medium within 15 h of incubation at 27°C. When using 5-isopropylhydantoin as substrate, N-carbamyl-valine was detected as the end product of the crude hydantoinase. Conditions leading to the isolation and conservation of a crude hydantoinase as well as its temperature and pH stability are described.     

Sporicidal activity of tertiary butyl hydroperoxide

Tertiary butyl hydroperoxide (t-BOOH) was found to be sporicidal for Bacillus megaterium ATCC19213. Sporicidal action was very temperature dependent, and the potency of t-BOOH increased about tenfold for each increase in temperature of 15 °C over the range from 30° to 70 °C. At still higher temperatures, heat and molar levels of t-BOOH were mutually potentiating for killing. Vegetative cells and germinated spores were some thousand times less resistant to t-BOOH than dormant spores. The order of resistance for spores was: Bacillus stearothermophilus ATCC7953 > Bacillus subtilis var. niger = Bacillus megaterium ATCC33729 > Bacillus megaterium ATCC19213. Killing was not enhanced by decoating and occurred without germination or loss of refractility of the spores. Spore resistance to t-BOOH was lower at more acid pH values and was decreased also by demineralization. Spores could be protected by the chelator o-phenanthroline, especially in association with Fe²⁺. Overall, it seemed that killing was associated with nonmetabolic formation of alkyl peroxyl radicals, which are thought to be responsible for killing of vegetative cells by organic hydroperoxides.Abbreviation A-BOOH tertiary butyl hydroperoxide     

Aerobic biodegradation of nonylphenol by cold-adapted bacteria

Three strains capable of mineralizing nonylphenol as sole carbon source were isolated from a sample of contaminated soil and characterized as two Pseudomonas spp. and a Stenotrophomonas sp. The two Pseudomonas spp. expressed characteristics typical of psychrophiles growing optimally of 10 °C and capable of growing at 0 °C. The Stenotrophomonas sp. was more likely psychrotrophic because it had an optimal temperature between 14 and 22 °C although it was not capable of growing at 4 °C. At 14 °C, one of the Pseudomonas spp. exhibited the highest rate of degradation of nonylphenol (4.4 mg l^–1 d^–1), when compared with axenic or mixed cultures of the isolates. This study represents, to the best of our knowledge, the first reported case of cold-adapted microorganisms capable of mineralizing nonylphenol.     

Temperature shiftup suppresses synthesis of extracellular proteins inBacillus megaterium

The effects of temperature shifts on the synthesis of an extracellular proteinase and extracellular proteins inBacillus megaterium were studied. A shiftdown (42° to 28°C) brought about an immediate increase of proteinase synthesis by 70%. A shiftup (28° to 42°C) caused a temporary suppression of enzyme formation, which was, at least partially, owing to the inhibition of its mRNA synthesis. The shiftup also brought about a temporary decrease of excretion of all extracellular proteins.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30 °C, compared with 53 °C for EcTrpase, and loses activity rapidly (t_1/2 ∼ 30 min) when incubated at 50 °C, while EcTrpase is stable up to 65 °C. PpTrpase retains complete activity when incubated more than 3 h at 0 °C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100 MPa (986 atm), while EcTrpase exhibits only about 10% activity at 100 MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.     

Change in chemical composition of membrane of Bacillus caldotenax after shifting the growth temperature

Membranes from Bacillus caldotenax contain neutral lipids and phospholipids such as phosphatidylethanolamine, phosphatidyl glycerol and cardiolipin. Each of the lipids has almost the same fatty acid composition. When the growth temperature decreases, not only the fatty acid composition but also the lipid composition changes such that the membrane fluidity increases, and the composition of membrane-bound proteins also changes. On shifting the growth temperature from 65° to 45°C, the bacterium grows immediately with a doubling time at 45°C, but the compositions of proteins and lipids in membranes gradually change and reach the compositions typical of cells growing at 45°C one doubling time after the temperature shift, respectively. It is concluded that the change in chemical composition of membrane of the bacterium on the temperature shift from 65° to 45°C is not prerequisite for growth at 45°C.     

<Emphasis Type="Italic">Thermobaculum terrenum</Emphasis> gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65–92 °C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 °C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1^T (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).     

Isolation and characterization of thermophilic,alkaliphilic, cellulose‐degrading bacillus thermoalcaliphilus sp. nov. from termite (odontotermes obesus) mound soil of a semiarid area

A new heterotrophic, thermophilic, alkaliphilic, facultatively anaerobic, cellulose‐degrading bacterium strain STS1 was isolated from mound soils infested with the higher termite Odontotermes obesus in the semiarid ecosystem of Delhi (India). The gram‐positive, spore‐forming, catalase‐positive Bacillus sp. grew on natural and raw celluloses. The taxonomic position of the organism was investigated. The guanine plus cytosine content of the isolate was found to be 48.6 mol% (melting temperature profile). Addition of peptone or yeast extract stimulated growth. The isolate did not grow on silica gel plates or on agar media in which the agar was the sole source of carbon and energy. The high growth temperature of 70°C and the pH of 9.0 are characteristic of this species. The role of this bacterium in the semiarid ecosystem is discussed. Because of its high optimum temperature and high optimum pH for growth, the name Bacillus thermoalcaliphilus is proposed.     

A thermostable extracellular xylanase from alkalophilic Bacillus sp. NG-27

Summary An alkalophilicBacillus sp. NG-27, which produced a thermostable xylanase was isolated from soil. The xylanase was active in the temperature range of 40–90°C with a temperature optimum at 70°C. It had a half life of 75 min at 70°C.     

Effects of temperature and medium composition on the ethanol tolerance ofBacillus stearothermophilus LLD-15

The effects of temperature (60°–70°C) and medium composition (complex and defined) on ethanol tolerance ofBacillus stearothermophillus LLD-15, an L-lactate dehydrogenase mutant have been determined in shake flasks under aerobic conditions. In all cases, there was complete inhibition of growth in the presence of 6%v/v ethanol.B. stearothermophillus LLD-15 was found to be less tolerant to ethanol at 70°C than at 60°C and also less tolerant to ethanol in a defined medium than in a complex medium.