Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu²⁺. The Michaelis constant (K_m) and V_max for dimethoate were 1.25 mM and 292 μmol min⁻¹ mg of protein⁻¹, respectively.     

Resolution of Distinct Membrane-Bound Enzymes from Enterobacter cloacae SLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ~600 kDa. The individual subunit sizes are ~100 kDa (α), ~55 kDa (β), and ~36 kDa (γ), with a predicted overall subunit composition of α₃β₃γ₃. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent K_m for selenate was determined to be ~2 mM, with an observed V_max of 500 nmol SeO₄²⁻ min⁻¹ mg⁻¹ (k_cat, ~5.0 s⁻¹). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (k _cat/K _m) of Coxyn A was 366 mg ml⁻¹ s⁻¹ on beechwood xylan, and the catalytic efficiency (k _cat/K _m) of Coxyl A was 2253 mM⁻¹ s⁻¹ on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Production, Purification, and Characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K_m of 7.9 mg/ml and an apparent V_max of 305 μmol · min^-1 · mg of protein^-1. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The K_m value toward pullulan was 2 mg ml⁻¹, with a V_max of 109 U mg⁻¹. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.     

Purification and Properties of a Xylan-Binding Endoxylanase from Alkaliphilic Bacillus sp. Strain K-1

An alkaliphilic bacterium, Bacillus sp. strain K-1, produces extracellular xylanolytic enzymes such as xylanases, β-xylosidase, arabinofuranosidase, and acetyl esterase when grown in xylan medium. One of the extracellular xylanases that is stable in an alkaline state was purified to homogeneity by affinity adsorption-desorption on insoluble xylan. The enzyme bound to insoluble xylan but not to crystalline cellulose. The molecular mass of the purified xylan-binding xylanase was estimated to be approximately 23 kDa. The enzyme was stable at alkaline pHs up to 12. The optimum temperature and optimum pH of the enzyme activity were 60°C and 5.5, respectively. Metal ions such as Fe²⁺, Ca²⁺, and Mg²⁺ greatly increased the xylanase activity, whereas Mn²⁺ strongly inhibited it. We also demonstrated that the enzyme could hydrolyze the raw lignocellulosic substances effectively. The enzymatic products of xylan hydrolysis were a series of short-chain xylooligosaccharides, indicating that the enzyme was an endoxylanase.     

Characterization of an Acetyl Xylan Esterase from the Anaerobic Fungus Orpinomyces sp. Strain PC-2

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K_m of 0.9 mM and a V_max of 785 μmol min⁻¹ mg⁻¹. It had temperature and pH optima of 30°C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), K_m (0.041 mM), and V_max (2.008 µmol.mg protein ⁻¹.min⁻¹) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L⁻¹ vs 36 mg.L⁻¹) and total enzyme activity increased about 5 folds (1900 IU.L⁻¹ vs 367 IU.L⁻¹) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.     

Thermophilic, reversible gamma-resorcylate decarboxylase from Rhizobium sp. strain MTP-10005: purification, molecular characterization, and expression

We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of k_cat/K_m (mΜ⁻¹·s⁻¹) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.     

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: K_m values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and k_cat/K_m values of 25.83 mM⁻¹ s⁻¹, 7.63 mM⁻¹ s⁻¹, 3.83 mM⁻¹ s⁻¹ and 3.75 mM⁻¹ s⁻¹, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.     

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO₂ fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min⁻¹mg protein⁻¹. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP_i. The enzyme was purified 95-fold to a specific activity of 18 μmol min⁻¹ mg protein⁻¹ from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO₂ fixation in this archaeon and possibly other members of the Sulfolobaceae family.     

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

Decreasing the Level of Ethyl Acetate in Ethanolic Fermentation Broths of Escherichia coli KO11 by Expression of Pseudomonas putida estZ Esterase

During the fermentation of sugars to ethanol relatively high levels of an undesirable coproduct, ethyl acetate, are also produced. With ethanologenic Escherichia coli strain KO11 as the biocatalyst, the level of ethyl acetate in beer containing 4.8% ethanol was 192 mg liter⁻¹. Although the E. coli genome encodes several proteins with esterase activity, neither wild-type strains nor KO11 contained significant ethyl acetate esterase activity. A simple method was developed to rapidly screen bacterial colonies for the presence of esterases which hydrolyze ethyl acetate based on pH change. This method allowed identification of Pseudomonas putida NRRL B-18435 as a source of this activity and the cloning of a new esterase gene, estZ. Recombinant EstZ esterase was purified to near homogeneity and characterized. It belongs to family IV of lipolytic enzymes and contains the conserved catalytic triad of serine, aspartic acid, and histidine. As expected, this serine esterase was inhibited by phenylmethylsulfonyl fluoride and the histidine reagent diethylpyrocarbonate. The native and subunit molecular weights of the recombinant protein were 36,000, indicating that the enzyme exists as a monomer. By using α-naphthyl acetate as a model substrate, optimal activity was observed at pH 7.5 and 40°C. The K_m and V_max for α-naphthyl acetate were 18 μM and 48.1 μmol·min⁻¹·mg of protein⁻¹, respectively. Among the aliphatic esters tested, the highest activity was obtained with propyl acetate (96 μmol·min⁻¹·mg of protein⁻¹), followed by ethyl acetate (66 μmol·min⁻¹·mg of protein⁻¹). Expression of estZ in E. coli KO11 reduced the concentration of ethyl acetate in fermentation broth (4.8% ethanol) to less than 20 mg liter⁻¹.