Geobacillus Thermodenitrificans NG80-2 Research Articles

Facultative anaerobic conversion of lignocellulose biomass to new bioemulsifier by thermophilic Geobacillus thermodenitrificans NG80-2.

Heavy oil has hindered crude oil exploitation and pollution remediation due to its high density and viscosity. Bioemulsifiers efficiently facilitate the formation and stabilization of oil-in-water emulsions in low concentrations thus eliminating the above bottleneck. Despite their potential benefits, various obstacles had still impeded the practical applications of bioemulsifiers, including high purification costs and poor adaptability to extreme environments such as high temperature and oxygen deficiency. Herein, thermophilic facultative anaerobic Geobacillus thermodenitrificans NG80-2 was proved capable of emulsifying heavy oils and reducing their viscosity. An exocelluar bioemulsifier could be produced by NG80-2 using low-cost lignocellulose components as carbon sources even under anaerobic condition. The purified bioemulsifier was proved to be polysaccharide-protein complexes, and both components contributed to its emulsifying capability. In addition, it displayed excellent stress tolerance over wide ranges of temperatures, salinities, and pHs. Meanwhile, the bioemulsifier significantly improved oil recovery and degradation efficiency. An eps gene cluster for polysaccharide biosynthesis and genes for the covalently bonded proteins was further certificated. Therefore, the bioemulsifier produced by G. thermodenitrificans NG80-2 has immense potential for applications in bioremediation and EOR, and its biosynthesis pathway revealed here provides a theoretical basis for increasing bioemulsifier output.

Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0-8.5, has highest activity at 60°C and is 100% stable when incubated for 16h at 55°C. DTT, β-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD Ap and NTD-fused ntdSOD Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD Ap . To the best of our knowledge, Mn-rec ntdSOD Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD Ap and ntdSOD Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.

Synergistic hydrolysis of xylan using novel xylanases, β-xylosidases, and an α-L-arabinofuranosidase from Geobacillus thermodenitrificans NG80-2.

Lignocellulosic biomass from various types of wood has become a renewable resource for production of biofuels and biobased chemicals. Because xylan is the major component of wood hemicelluloses, highly efficient enzymes to enhance xylan hydrolysis can improve the use of lignocellulosic biomass. In this study, a xylanolytic gene cluster was identified from the crude oil-degrading thermophilic strain Geobacillus thermodenitrificans NG80-2. The enzymes involved in xylan hydrolysis, which include two xylanases (XynA1, XynA2), three β-xylosidases (XynB1, XynB2, XynB3), and one α-L-arabinofuranosidase (AbfA), have many unique features, such as high pH tolerance, high thermostability, and a broad substrate range. The three β-xylosidases were highly resistant to inhibition by product (xylose) accumulation. Moreover, the combination of xylanase, β-xylosidase, and α-L-arabinofuranosidase exhibited the largest synergistic action on xylan degradation (XynA2, XynB1, and AbfA on oat spelt or beechwood xylan; XynA2, XynB3, and AbfA on birchwood xylan). We have demonstrated that the proposed enzymatic cocktail almost completely converts complex xylan to xylose and arabinofuranose and has great potential for use in the conversion of plant biomass into biofuels and biochemicals.

DFT Study on Enzyme Turnover Including Proton and Electron Transfers of Copper-Containing Nitrite Reductase.

The reaction mechanism of copper-containing nitrite reductase (CuNiR) has been proposed to include two important events, an intramolecular electron transfer and a proton transfer. The two events have been suggested to be coupled, but the order of these events is currently under debate. We investigated the entire enzyme reaction mechanism of nitrite reduction at the T2 Cu site in thermophilic Geobacillus CuNiR from Geobacillus thermodenitrificans NG80-2 (GtNiR) using density functional theory calculations. We found significant conformational changes of His ligands coordinated to the T2 Cu site upon nitrite binding during the catalytic reaction. The reduction potentials and pKa values calculated for the relevant protonation and reduction states show two possible routes, A and B. Reduction of the T2 Cu site in the resting state is followed by endothermic nitrite binding in route A, while exothermic nitrite binding occurs prior to reduction of the T2 Cu site in route B. We concluded that our results support the random-sequential mechanism rather than the ordered mechanism.

Improving the thermostability and stress tolerance of an archaeon hyperthermophilic superoxide dismutase by fusion with a unique N-terminal domain.

The superoxide dismutase from the archaeon Sulfolobus solfataricus (SODSs) is a well-studied hyperthermophilic SOD with crystal structure and possible thermostability factors characterized. Previously, we discovered an N-terminal domain (NTD) in a thermophilic SOD from Geobacillus thermodenitrificans NG80-2 which confers heat resistance on homologous mesophilic SODs. The present study therefore aimed to further improve the thermostability and stress tolerance of SODSs via fusion with this NTD. The recombinant protein, rSODSs, exhibited improved thermophilicity, higher working temperature, improved thermostability, broader pH stability, and enhanced tolerance to inhibitors and organic media than SODSs without any alterations in its oligomerization state. These results suggest that the NTD is an excellent candidate for improving stability of both mesophilic and thermophilic SOD from either bacteria or archaea via simple genetic manipulation. Therefore, this study provides a general, feasible and highly useful strategy for generating extremely thermostable SODs for industrial applications. Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1854-9) contains supplementary material, which is available to authorized users.

Cloning, expression, and characterization of a thermostable l-arginase from Geobacillus thermodenitrificans NG80-2 for l-ornithine production.

Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3U/mg. The molecular mass of the l-arginase was approximately 33.0kDa as estimated by SDS-PAGE and 192.0kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80°C, and the protein was stable at 40 and 50°C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9mM and 3.8mM(-1) s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9g/L, and the molar yield was 97.2%.

A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs.

Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70°C and high thermostability over a broad range of temperatures (20–80°C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30°C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55°C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs.

A novel type I thermostable pullulanase isolated from a thermophilic starch enrichment culture

In this paper we report identification, cloning and characterization of a novel thermostable pullulanase type I. Pullulanase AmyA1 was detected in a sample of extracellular proteins of thermophilic enrichment culture, growing on starch. The zone of enzymatic activity in zymogram was aligned with the corresponding band on the equivalent gel without substrate. The band was excised from SDS/polyacrylamide gel and subjected to liquid chromatography/mass spectrometry (LC/MS) analysis. LC/MS-based analysis identified thermostable pullulanases, homologues to type I pullulanases of Geobacillus thermodenitrificans NG80-2 and Geobacillus sp. G11MC16. Nucleotide sequences of these two pullulanases were used for design of primers for PCR with DNA from enrichment culture, leaded to 2181 bp PCR product, coding a 726 amino acids protein, named pullulanase AmyA1. Molecular weight of AmyA1 was calculated to be 81.7 kDa. AmyA1 was cloned and expressed in Escherichia coli. Recombinant pullulanase was purified by two chromatographic separation steps. Pullulanase AmyA1 was active against pullulan, glycogen and soluble starch. It was active in the temperature range of 4–95°C, optimum temperature was determined to be 60°C. The highest activity of the recombinant pullulanase was observed at pH 6. Divalent cations Mg2+ and Mn2+ as well as dithiothreitol, Brij 35 and Brij 58 had a stimulating effect on the enzymatic activity. Pullulanase AmyA1 was stable during incubation in the presence of 4 M urea. After removal of the His-tag, addition of Ca2+ stimulated activity of the enzyme suggesting the native pullulanase activity to be dependent on Ca2+. Thermostability of AmyA1 was not enhanced by the addition of Ca2+.

Crystallization and preliminary X-ray characterization of an NAD(P)-dependent butanol dehydrogenase A from Geobacillus thermodenitrificans NG80-2.

Geobacillus thermodenitrificans NG80-2 encodes two long-chain NAD(P)-dependent alcohol dehydrogenases, gtADH1 and gtADH2, in the terminal oxidation pathway of long-chain n-alkanes for the conversion of long-chain alkyl alcohols to their corresponding aldehydes. Both gtADH1 and gtADH2 are thermostable enzymes and oxidize long-chain alkyl alcohols up to at least C(30). In order to understand the structural basis for their role in long-chain alkane degradation, we have crystallized gtADH2. Single, colourless crystals were obtained from a recombinant preparation of ADH2 overexpressed in Escherichia coli. The crystals belong to space group C222(1), with unit-cell parameters a = 56.0, b = 99.6, c = 123.1 Å. Diffraction data were collected in-house to 1.79 Å resolution. The crystals contain one monomer in the asymmetric unit, with a V(M) value of 2.17 Å(3) Da(-1) and an estimated solvent content of 43%.

Biochemical and conformational characterization of a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2

In order to search for valuable and extremely thermo-stable enzymes that could be used in the protein hydrolysis industry, the gene corresponding to a leucine aminopeptidase from Geobacillus thermodenitrificans NG80-2 (GtLAP) was cloned and expressed in E. coli. The recombinant enzyme was purified, and its characteristics were examined. Meanwhile, potential applications of GtLAP in the hydrolysis of anchovy proteins were also investigated. GtLAP was overexpressed in IPTG-induced E. coli BL21 (pET28a-LAP) as a soluble protein, and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 125 ± 8.75 U/mg proteins. The molecular mass of GtLAP was estimated to be 55 kDa by SDS-PAGE analysis. The optimal reaction temperature and pH of GtLAP were 70 °C and 8.0, respectively. Under optimal conditions, GtLAP showed a marked preference for Leu-p-nitroanilide, followed by Met- and Phe-derivatives. Activity of GtLAP was strongly stimulated by Ni²⁺ ions, but was strongly inhibited by Hg²⁺. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of GtLAP to various extents and further induce changes in enzymatic activity. Results of hydrolytic experiment showed that combining GtLAP with endogenous enzymes could significantly increase the degree of hydrolysis to anchovy proteins and concentrations of free amino acids in hydrolysates. In this regard, GtLAP could potentially be used in the protein hydrolysis industry.

Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitrificans NG80-2

The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C2) to melissic acid (C30). The most preferred substrates were capric acid (C10) for Facl1 and palmitic acid (C16) for Facl2, respectively. Both enzymes had an optimal temperature of 60°C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C30 has been demonstrated in bacteria.

Engineering of LadA for enhanced hexadecane oxidation using random- and site-directed mutagenesis

LadA, a monooxygenase catalyzing the oxidation of n-alkanes to 1-alkanols, is the key enzyme for the degradation of long-chain alkanes (C(15)-C(36)) in Geobacillus thermodenitrificans NG80-2. In this study, random- and site-directed mutagenesis were performed to enhance the activity of the enzyme. By screening 7,500 clones from random-mutant libraries for enhanced hexadecane hydroxylation activity, three mutants were obtained: A102D, L320V, and F146C/N376I. By performing saturation site-directed mutagenesis at the 102, 320, 146, and 376 sites, six more mutants (A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, and F146N/N376I) were generated. Kinetic studies showed that the hydroxylation activity of purified LadA mutants on hexadecane was 2-3.4-fold higher than that of the wild-type enzyme, with the activity of F146N/N376I being the highest. Effects of the mutations on optimum temperature, pH, and heat stability of LadA were also investigated. A complementary study showed that Pseudomonas fluorescens KOB2Δ1 strains expressing the LadA mutants grew more rapidly with hexadecane than the strain expressing wild-type LadA, confirming the enhanced activity of LadA mutants in vivo. Structural changes resulting from the mutations were analyzed and the correlation between structural changes and enzyme activity was discussed. The mutants generated in this study are potentially useful for the treatment of environmental oil pollution and in other bioconversion processes.

Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71

The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al(3+) and Cu(2+) but strongly inhibited by Hg(2+). The enzyme follows Michaelis-Menten kinetics, with K(m) and V(max) values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.

Proteomics analysis of aromatic catabolic pathways in thermophilic Geobacillus thermodenitrificans NG80-2

Geobacillus thermodenitrificans NG80-2 is a crude oil-degrading thermophilic bacterium isolated from an oil reservoir in China. In this study, the gene clusters and pathways for the degradation of benzoate (via benzoyl-CoA), phenylacetate (via phenylacetyl-CoA), 4-hydroxyphenylacetate (via 3,4-dihydroxyphenylacetate) and anthranilate (via 3-hydroxyanthranilate) were confirmed using combined in silico analysis and proteomics approaches. It was observed that synthesis of the enzymes catalyzing initial activation, ring oxidation and ring cleavage reactions were generally induced specifically by their respective substrates, while many of the enzymes catalyzing downstream reactions exhibited broader substrate specificities. Novel genes encoding benzoyl-CoA epoxidase and 3,4-dihydroxyphenylacetate 2,3-dioxygenase, and a paaX homologue that serves as a positive regulator of benzoate degradation were proposed. Downregulation of the glycolysis pathway, along with upregulation of the gluconeogenesis pathway and the glyoxylate bypass (phenylacetate) were detected in association with the utilization of the aromatics. This novel proteomics approach confirmed the presence of multiple metabolic pathways for aromatic compounds in NG80-2, which is highly advantageous to the survival of this thermophilic bacterium under reservoir conditions.

Characterization of a broad-range aldehyde dehydrogenase involved in alkane degradation in Geobacillus thermodenitrificans NG80-2

An aldehyde dehydrogenase (ALDH) involved in alkane degradation in crude oil-degrading Geobacillus thermodenitrificans NG80-2 was characterized in vitro. The ALDH was expressed heterologously in Escherichia coli and purified as a His-tagged homotetrameric protein with a subunit of 57 kDa based on SDS-PAGE and Native-PAGE analysis. The purified ALDH-oxidized alkyl aldehydes ranging from formaldehyde (C₁) to eicosanoic aldehyde (C₂₀) with the highest activity on C₁. It also oxidized several aromatic aldehydes including benzaldehyde, phenylacetaldehyde, o-chloro-benzaldehyde and o-phthalaldehyde. The ALDH uses only NAD(+) as the cofactor, and has no reductive activity on acetate or hexadecanoic acid. Therefore, it is an irreversible NAD(+)-dependent aldehyde dehydrogenase. Kinetic parameters, temperature and pH optimum of the enzyme, and effects of metal ions, EDTA and Triton X-100 on the enzyme activity were investigated. Physiological roles of the ALDH for the survival of NG80-2 in oil reservoirs are discussed.

Characterization of the anthranilate degradation pathway in Geobacillus thermodenitrificans NG80-2

Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH(2)-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH(2)) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated.

Identification of a Novel Two-Peptide Lantibiotic, Lichenicidin, following Rational Genome Mining for LanM Proteins

Lantibiotics are ribosomally synthesized peptide antimicrobials which contain considerable posttranslational modifications. Given their usually broad host range and their highly stable structures, there have been renewed attempts to identify and characterize novel members of the lantibiotic family in recent years. The increasing availability of bacterial genome sequences means that in addition to traditional microbiological approaches, in silico screening strategies may now be employed to the same end. Taking advantage of the highly conserved nature of lantibiotic biosynthetic enzymes, we screened publicly available microbial genome sequences for genes encoding LanM proteins, which are required for the posttranslational modification of type 2 lantibiotics. By using this approach, 89 LanM homologs, including 61 in strains not known to be lantibiotic producers, were identified. Of these strains, five (Streptococcus pneumoniae SP23-BS72, Bacillus licheniformis ATCC 14580, Anabaena variabilis ATCC 29413, Geobacillus thermodenitrificans NG80-2, and Herpetosiphon aurantiacus ATCC 23779) were subjected to a more detailed bioinformatic analysis. Four of the strains possessed genes potentially encoding a structural peptide in close proximity to the lanM determinants, while two, S. pneumoniae SP23-BS72 and B. licheniformis ATCC 14580, possess two potential structural genes. The B. licheniformis strain was selected for a proof-of-concept exercise, which established that a two-peptide lantibiotic, lichenicidin, which exhibits antimicrobial activity against all Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococcus strains tested, was indeed produced, thereby confirming the benefits of such a bioinformatic approach when screening for novel lantibiotic producers.

Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2

Two alkyl alcohol dehydrogenase (ADH) genes from the long-chain alkane-degrading strain Geobacillus thermodenitrificans NG80-2 were characterized in vitro. ADH1 and ADH2 were prepared heterologously in Escherichia coli as a homooctameric and a homodimeric protein, respectively. Both ADHs can oxidize a broad range of alkyl alcohols up to at least C(30), as well as 1,3-propanediol and acetaldehyde. ADH1 also oxidizes glycerol, and ADH2 oxidizes isopropyl alcohol, isoamylol, acetone, octanal and decanal. The best substrate is ethanol for ADH1 and 1-octanol for ADH2. For both ADHs, the optimum assay condition is at 60 degrees C and pH 8.0, and both NAD and NADP can be used as the cofactor. Sequence analysis reveals that ADH1 and ADH2 belong to the Fe-containing/activated long-chain ADHs. However, the two enzymes contain neither Fe nor other metals, and Fe is not required for the activity, suggesting a new type of ADH. The ADHs characterized here are potentially useful in crude oil bioremediation and other bioconversion processes.

Thenosgene cluster from gram-positive bacteriumGeobacillus thermodenitrificansNG80-2 and functional characterization of the recombinant NosZ

The nos gene cluster encoding the activity of nitrous oxide reductase (N2OR) for the final step of the denitrification pathway has been well studied in gram-negative bacteria. Our previous study on the genome of Geobacillus thermodenitrificans NG80-2 revealed the presence of the nos gene cluster in this gram-positive bacterium. In this follow-up study, the nos gene cluster of G. thermodenitrificans NG80-2 was further analyzed and compared with those of other origins. The structural gene nosZ was heterologously expressed in Escherichia coli and the product was purified as a His-tagged fusion protein. The recombinant NosZ as prepared showed detectable N2OR activity, and the activity was enhanced by preincubation of the protein under argon and with copper compounds. The recombinant NosZ contains 2.5 atoms of copper per dimer and exhibits weak spectral features in the visible range, indicating that spontaneous incorporation of copper compounds into the NG80-2 NosZ can result in some but not full activity of the authentic NG80-2 N2OR. The enzymatic properties of the NosZ were also investigated. This is the first functional characterization of nosZ gene from gram-positive bacteria. This study indicates that the molecular mechanism for N2O reduction is conserved between gram-negative and gram-positive bacteria.