A specific amino acid residue in the catalytic site of dandelion polyphenol oxidases acts as ‘selector’ for substrate specificity

Key message

Successful site-directed mutagenesis combined with in silico modeling and docking studies for the first time offers experimental proof of the role of the ‘substrate selector’ residue in plant polyphenol oxidases.

Abstract

The plant and fungi enzymes responsible for tissue browning are called polyphenol oxidases (PPOs). In plants, PPOs often occur as families of isoenzymes which are differentially expressed, but little is known about their physiological roles or natural substrates. In a recent study that explored these structure–function relationships, the eleven known dandelion (Taraxacum officinale) PPOs were shown to separate into two different phylogenetic groups differing in catalytic cavity architecture, kinetic parameters, and substrate range. The same study proposed that the PPOs’ substrate specificity is controlled by one specific amino acid residue positioned at the entrance to the catalytic site: whereas group 1 dandelion PPOs possess a hydrophobic isoleucine (I) at position H_B2+1, group 2 PPOs exhibit a larger, positively charged arginine (R). However, this suggestion was only based on bioinformatic analyses, not experiments. To experimentally investigate this hypothesis, we converted group 1 ToPPO-2 and group 2 ToPPO-6 into PPO-2-I₂₄₄R and PPO-6-R₂₅₄I, respectively, and expressed them in E. coli. By performing detailed kinetic characterization and in silico docking studies, we found that replacing this single amino acid significantly changed the PPO’s substrate specificity. Our findings therefore proof the role of the ‘substrate selector’ in plant PPOs.

     

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency

Background and Aims Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress.Methods Photosynthesis (A, F_v/F_m, ΦPSII, q_N, q_P, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense ‘Milvus’) under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m²s^–1) or 0–10 µm methyl viologen. Foliar protein content and oxidation state were also determined.Key Results Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses.Conclusions The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.     

Site-directed mutagenesis of a tetrameric dandelion polyphenol oxidase (PPO-6) reveals the site of subunit interaction

Polyphenol oxidases (PPOs) catalyze the oxidation of ortho-diphenols to the corresponding quinones (EC 1.10.3.1). In plants PPOs appear in gene families, and the corresponding isoenzymes are located to the thylakoid lumen of chloroplasts. Although plant PPOs are often discussed with regard to their role in defense reactions, a common physiological function has not yet been defined. We analyzed a tetrameric PPO isoenzyme (PPO-6) from dandelion (Taraxacum officinale) heterologously expressed in Escherichia coli, and found it to display cooperativity in catalysis, a phenomenon that has rarely been shown for plant PPOs previously. The identification of a surface-exposed cysteine (197) through molecular modeling followed by site-directed mutagenesis proved this amino acid residue to stabilize the tetramer via a disulfide linkage. The C197S-mutein still forms a tetrameric structure but shows impaired enzymatic efficiency and cooperativity and a reduction in stability. These findings indicate that oligomerization may be a physiological requirement for PPO-6 stability and function in vivo and raise new questions regarding distinct functions for specific PPO isoenzymes in plants.     

CotA,a Multicopper Oxidase from Bacillus pumilus WH4, Exhibits Manganese-Oxidase Activity

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl₂. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The K_m, V_max and k_cat values towards Mn(II) were 14.85±1.17 mM, 3.01×10⁻⁶±0.21 M·min⁻¹ and 0.32±0.02 s⁻¹, respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.     

Purification and Characterization of a Secreted Laccase of Gaeumannomyces graminis var. tritici

We purified a secreted fungal laccase from filtrates of Gaeumannomyces graminis var. tritici cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphenol (K_m = 2.6 × 10⁻⁵ ± 7 × 10⁻⁶ M), catechol (K_m = 2.5 × 10⁻⁴ ± 1 × 10⁻⁵ M), pyrogallol (K_m = 3.1 × 10⁻⁴ ± 4 × 10⁻⁵ M), and guaiacol (K_m = 5.1 × 10⁻⁴ ± 2 × 10⁻⁵ M). In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen’s hyphae and/or in lignin depolymerization in its infected plant host.     

Physiological and biochemical response of different resistant alfalfa cultivars against thrips damage

To investigate physiological and biochemical changes of thrips-resistant alfalfa (Medicago sativa L. cv. Gan-nong No. 9), we aimed at clarifying the response mechanisms of alfalfa against thrips. Medicago sativa L. cv. including thrips-resistant Gan-nong No.9 (G9), thrips-susceptible Gan-nong No.3 (G3) and highly thrips-susceptible WL363HQ (363) were infested with different thrips densities (3, 5, 7 and 9-thrips per branch). The quantitative change in specific nutrients, secondary metabolites, defensive and antioxidant enzymes were measured at seedling stage of the three alfalfa cultivars. The results showed that with the increase of thrips densities, the damage indices, SS, Pro, flavonoids, tannin and H₂O₂ in G9, G3 and 363 were significantly increased, but PPO and SOD significantly reduced, compared with CK. Furthermore, the tannin and lignin contents of G9 were significantly higher compared to 363, but SP content was significantly lower than G3 and H₂O₂ content which was further significantly less compared to 363. Correlation analysis observed that the damage index of the three alfalfa cultivars showed a significant positive association with SS, Pro, flavone, tannin, and H₂O₂ (P < 0.01), while damage index and DW, Chl (a, b, a + b), PPO and SOD showed a significant negative correlation (P < 0.01). Based on principal component comprehensive evaluation, the 5-thrips adults per branch were the critical inoculation threshold for G9 against thrips injury because the score was – 0.048. These results revealed that thrips damage significantly increased the contents of SS, Pro, flavonoids, tannins and H₂O₂, as well as significantly declined the activities of PPO and SOD in the three cultivars (P < 0.05), moreover, thrips-resistant G9 markedly accumulated lignin content, POD and CAT activity, inhibited Chl (a + b, b) and SP biosynthesis to resist thrips damage.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii

Type A glandular trichomes of the wild potato (Solanum berthaultii Hawkes) entrap insects by rapidly polymerizing the trichome contents after breakage by insect contact. Polymerization of trichome exudate appears to be driven by a soluble polyphenol oxidase (PPO). PPO constitutes up to 70% of the protein in individually collected trichomes and reaches a concentration approaching 200 μm in these organs. Trichome PPO has been purified and shown to be a monomeric copper metalloprotein with an isoelectric point of 5.5, possessing only o-diphenol oxygen oxido-reductase activity, and is larger than most other reported PPOs, with relative molecular weight of 59,000. Chlorogenic and caffeic acid were the most readily oxidized of 14 phenolic substrates tested. Polyclonal antibodies raised against the relative molecular weight 59,000 S. berthaultii trichome PPO were used to show that S. tuberosum L. trichomes express low levels of a cross-reactive protein that lacks detectable PPO activity.     

Comparison of polyphenol oxidase expression in glandular trichomes of solanum and lycopersicon species

Tetralobulate glandular trichomes are present on the foliage of many solanaceous species. Resistance of many of these species to insects is conditioned by the ability of trichomes to rupture upon contact and to rapidly polymerize their contents, resulting in entrapment of insects in hardened trichome exudate. In the wild potato, Solanum berthaultii, polymerization of trichome exudate is initiated by a soluble M_r 59,000 polyphenol oxidase (PPO), which is a dominant protein constituent of the organ. PPOs, although ubiquitous in angiosperms, typically display great heterogeneity in molecular weight and are found at low levels in plant cells. Because of the unusually high accumulation and tissue-specific expression of the M_r 59,000 PPO in S. berthaultii glandular trichomes, we analyzed trichome proteins of a number of Lycopersicon and Solanum species to assess the extent to which possession of the M_r 59,000 PPO is conserved. Trichomes were collected manually and examined for PPO activity, immuno-cross-reactivity with S. berthaultiiM_r 59,000 PPO, and protein content. In addition, N-terminal amino acid sequences were obtained for five trichome PPOs. All species analyzed possessed trichome PPOs similar in structure and level of expression to that of S. berthaultii. The relationship between sequences and structures of these conserved PPOs and the variable PPOs of leaf is discussed.     

Exo-β-glucanases in yeast

1. A number of yeast species were examined for the presence of β-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher β-(1→3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-β-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-β-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-β-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5·5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-β-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-δ-lactone gave the same value of K_i when tested with either substrate; (iii) quantitative application of the `mixed-substrate' method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-β-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and K_m values with laminarin and pustulan differed markedly. Comparison of V_max. and K_m values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-β-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

Production by Streptomyces viridosporus T7A of an Enzyme Which Cleaves Aromatic Acids from Lignocellulose

The lignocellulose-degrading actinomycete Streptomyces viridosporus T7A produced an extracellular esterase when grown in a mineral salts-yeast extract medium. Extracellular esterase activity was first detected during the late stationary phase and typically followed the appearance of intracellular activity. When the organism was grown in lignocellulose-supplemented medium, esterase activity was not increased, but lignocellulose-esterified p-coumaric acid and vanillic acid were released into the medium. Polyacrylamide gels showed that several extracellular esterases differing in substrate specificity were produced. Ultrafiltration was used to concentrate the esterase prior to purification. Activity was recovered mostly in the molecular weight fraction between 10,000 and 100,000. Concentrated esterase was further purified by DEAE-Sepharose anion-exchange chromatography to a specific activity 11.82 times greater than that in the original supernatant. There were seven detectable esterase active proteins in the partially purified enzyme solution. Three were similar esterases that may be isoenzymes. The partially purified esterase had a pH optimum for activity of 9.0, a temperature optimum of 45 to 50°C, and a K_m and V_max of 0.030 mM and 0.097 μmol/min per ml, respectively, when p-nitrophenyl butyrate was the substrate. The enzyme was unstable above 40°C but retained activity when stored at 4 or −20°C. It lost some activity (20%) when lyophilized. Substrate specificity assays showed that it hydrolyzed ester linkages of p-nitrophenyl butyrate, α-naphthyl acetate, α-naphthyl butyrate, and lignocellulose. Vanillic and p-coumaric acids were identified as products released from lignocellulose. The enzyme is thought to be a component of the lignocellulose-degrading enzyme system of S. viridosporus.     

Thermostable Amylolytic Enzymes from a New Clostridium Isolate

A new Clostridium strain was isolated on starch at 60°C. Starch, pullulan, maltotriose, and maltose induced the synthesis of α-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of α-amylase and pullulanase into the culture broth; but also by a decrease of total activity. α-Amylase, pullulanase, and α-glucosidase were active in a broad temperature range (40 to 85°C) and displayed temperature optima for activity at 60 to 70°C. During incubation with starch under aerobic conditions at 75°C for 2 h, the activity of both enzymes decreased to only 90 or 80%. The apparent K_m values of α-amylase, pullulanase, and α-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively.     

A purified energy-converting hydrogenase from Thermoanaerobacter kivui demonstrates coupled H+-translocation and reduction in vitro

Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H₂. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H₂-dependent Fd reduction and Fd^2--dependent H₂ production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na⁺ for activity and did not translocate ²²Na⁺ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H⁺-translocating, H⁺-reducing enzyme.     

Seasonal variation in the antioxidant system of eastern white pine needles : evidence for thermal dependence

Antioxidant metabolites in eastern white pine (Pinus strobus L.) needles increased two- to fourfold from the summer to the winter season. Antioxidant enzymes in needle tissue increased between 2- and 122-fold during this same period. These seasonal changes were determined by monitoring ascorbate and glutathione concentrations and the activity of ascorbate peroxidase, glutathione reductase (GR), and superoxide dismutase. Levels of antioxidant metabolites and enzymes were observed always to be lowest during the summer, or active growing season, and highest during the winter, or dormant season. These data correlated well with the thermal kinetic window for purified GR obtained from summer needles. The minimum, apparent K_m,NADPH for two isoforms of GR (GR_A and GR_B) occurred at 5 and 10°C, respectively. The upper limit of the thermal kinetic window (200% of the minimum K_m) for GR_A and GR_B was 20 and 25°C, respectively, indicating that needle temperatures exceeding 25°C may result in impairment of antioxidant metabolism. The needle content and kinetic properties of GR, the increased activities of other enzymes, and the high substrate concentrations observed during the winter are consistent with the protective function this pathway may provide against photooxidative, winter injury.     

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690 ± 20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large (~1.6 MDa) complex that is not active. The apparent K_m values and catalytic efficiencies for the substrates pyruvate and ATP (at 80°C, pH 8.4) were 0.11 mM and 1.43 × 10⁴ mM⁻¹ · s⁻¹ and 0.39 mM and 3.40 × 10³ mM⁻¹ · s⁻¹, respectively. Maximal activity was measured at pH 9.0 (at 80°C) and at 90°C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k_cat/K_m = 32 (mM · s)⁻¹]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration (~5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

Collagenase Production by Nematode-Trapping Fungi

A number of species of nematode-trapping fungi, which capture and digest nematodes having keratin and collagen in their cuticles, were tested for the ability to produce extracellular collagenase and keratinase. Collagenase, which is active on ichthyocol, earthworm collagen, and procollagen from chicken embryo fibroblasts, was found in the growth medium of all tested species; keratinase was not found. The enzyme from Arthrobotrys amerospora was concentrated by precipitation with (NH₄)₂SO₄ and further purified by adsorption on collagen at 0°C. The collagenase was active over a pH range of 2.5 to 10.0. It was not inactivated by dialysis against ethylenediaminetetraacetic acid for 48 h or by the sulfhydryl group inhibitors N-ethylmaleimide and p-chloromercuribenzoate. The production of collagenase may aid the fungus to penetrate the cuticle of its prey.