Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, <Emphasis Type="Italic">Flavobacterium frigidimaris</Emphasis> KUC-1

An NAD⁺-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD⁺ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD⁺. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85°C and the most active at 70°C. The half-life time and k _cat value at 60°C were calculated to be 50 min and 27,400 min⁻¹, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k _cat/K _m at 10°C; 12,600 mM⁻¹ min⁻¹) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.     

Comparison of angiotensin converting enzyme-like activity in the Antarctic teleosts <Emphasis Type="Italic">Trematomus bernacchii</Emphasis> and <Emphasis Type="Italic">Chionodraco hamatus</Emphasis>

Biochemical parameters of the angiotensin converting enzyme-like activity (ACELA) in the gills of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii were characterized. Enzymatic activity was revealed following hydrolysis of a specific substrate of angiotensin-converting enzyme N-[3-(2-furyl)acryloyl]l-phenylalanyl-glycyl-glycine (FAPGG) and metabolites were separated by reverse phase HPLC analysis. The results showed similar Km values for the substrate FAPGG at 5°C for the two species with an increase of Km value for T. bernacchii at 25°C. The optimum pH value was 8.5 at 25°C and optimum chloride concentrations were about 300 mM. In T. bernacchii the optimum temperature for maximum enzyme activity was 50°C, while maximum activity in C. hamatus occurred at 35°C. Lisinopril was more efficient in inhibiting ACELA in C. hamatus with an I ₅₀ value of 16.83 ± 5.11 nM, compared to an I ₅₀ value of 30.66 ± 5.19 nM in T. bernacchii. In conclusion, it appears that some biochemical parameters of ACELA in C. hamatus differ from those in T. bernacchii, probably due to different ways that the enzyme adapts to the constantly cold temperatures of the animal’s environment.     

Expression of secondary alcohol dehydrogenase in methanogenic bacteria and purification of the F420-specific enzyme from Methanogenium thermophilum strain TCI

In four species of methanogens able to grow with secondary alcohols as hydrogen donors the expression and properties of secondary alcohol dehydrogenase (sec-ADH) were investigated. Cells grown with 2-propanol and CO₂ immediately started to oxidize secondary alcohols to ketones if transferred to new media. In the presence of H₂, such cells reduced ketones or aldehydes to alcohols. In the absence of H₂, aldehydes were dismutated (without growth) to primary alcohols and fatty acids. None of these reactions was catalyzed by cells grown with only H₂ and CO₂ at non-limiting concentration. This indicated an induction or derepression of sec-ADH by its substrate. Apparently, sec-ADH in all strains enabled not only the reduction of ketones or aldehydes, but also the dismutation of the latter. Sec-ADH was also expressed if strains were grown on H₂ and CO₂ in the presence of non-oxidizable, tertiary alcohols. Methanogenium thermophilum expressed sec-ADH even without added alcohol when H₂ became limiting. From this species, an F₄₂₀-specific sec-ADH was purified; the final gel filtration chromatography yielded a single protein peak that coincided with the activity. The enrichment was 12-fold, the activity recovery 26%. SDS polyacrylamide gel electrophoresis indicated that the enzyme was a homodimer with an apparent M _r of 79,000. At the pH optimum around 4.2, the specific activity for oxidation of 2-propanol (130 mM) and reduction of acetone (20 mM) was 176 and 110 mol/ min·mg, respectively (40°C). The apparent K _m for 2-propanol and acetone (with 15 M F₄₂₀) was 2.5 and 0.25 mM, respectively. Aldehydes also were reduced.Non-standard abbreviations ADH alcohol dehydrogenase - Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - F₄₂₀ N-(N-L-lactyl--L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mb. Methanobacterium - Mg. Methanogenium - Ms. Methanospirillum - OD₅₇₈ optical density at 578 nm - SDS sodium dodecyl sulfate     

<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V _max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD⁺ than with NADP⁺. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

Cloning,sequencing, overexpression and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-rhamnose isomerase from <Emphasis Type="Italic">Bacillus pallidus</Emphasis> Y25 for rare sugar production

The l-rhamnose isomerase gene (L -rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L -rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K _m) and catalytic efficiency (k _cat/K _m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 10⁵ M⁻¹ min⁻¹, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

A unique highly thermostable 2-phosphoglycerate forming glycerate kinase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>: gene cloning,expression and characterization

A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme (GK_ph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment and structural comparison it was assigned to group I of the trichotomy of GKs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO₂, and H₂, together with minor amounts of ethanol. The organism also generates H₂S in the presence of elemental sulfur (S⁰). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S⁰. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (~1.0 g-atom/subunit) and zinc (~1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent K_ms (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Archaeal alcohol dehydrogenase active at increased temperatures and in the presence of organic solvents

The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83°C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD⁺ as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t _1/2 = 42 min at 70°C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Purification and Properties of Primary and Secondary Alcohol Dehydrogenases from Thermoanaerobacter ethanolicus

Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent M_rs of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetrameric with apparently identical subunits and contain from 3.2 to 5.5 atoms of Zn per subunit. The two dehydrogenases are NADP dependent and reversibly convert ethanol and 1-propanol to the respective aldehydes. The V_m values with ethanol as a substrate are 45.6 μmol/min per mg for the primary alcohol dehydrogenase and 13 μmol/min per mg for the secondary alcohol dehydrogenase at pH 8.9 and 60°C. The primary enzyme oxidizes primary alcohols, including up to heptanol, at rates similar to that of ethanol. It is inactive with secondary alcohols. The secondary enzyme is inactive with 1-pentanol or longer chain alcohols. Its best substrate is 2-propanol, which is oxidized 15 times faster than ethanol. The secondary alcohol dehydrogenase is formed early during the growth cycle. It is stimulated by pyruvate and has a low K_m for acetaldehyde (44.8 mM) in comparison to that of the primary alcohol dehydrogenase (210 mM). The latter enzyme is formed late in the growth cycle. It is postulated that the secondary alcohol dehydrogenase is largely responsible for the formation of ethanol in fermentations of carbohydrates by T. ethanolicus.     

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn²⁺ significantly inhibited activity. The K _m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.     

Purification and Properties of an <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase from <Emphasis Type="Italic">Streptomyces cerradoensis</Emphasis>

An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The K_m and V_max values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg⁻¹ protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity was strongly inhibited by Cu²⁺ and Hg²⁺ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose.