Prophenoloxidase activation in the hemolymph of Sarcophaga bullata larvae

Phenoloxidase in the hemolymph of Sarcophaga bullata larvae is present as an inactive proenzyme form. Localization studies indicate that the majority of the prophenoloxidase is present in the plasma fraction whereas only a minor fraction (about 4%) is present in the cellular compartments (hemocytes). Inactive prophenoloxidase can be activated by zymosan, not by either endotoxin or laminarin. This activation process is inhibited by the serine protease inhibitors, benzamidine and p-nitrophenyl-p～-guanidobenzoate. Exogenously added proteases, such as chymotrypsin and subtilisin, also activated the prophenoloxidase in the whole hemolymph but failed to activate the partially purified proenzyme. However, an activating enzyme isolated from the larval cuticle, which exhibits trypsinlike specificity, activated the partially purified prophenoloxidase. Inhibition studies and activity measurements also revealed the presence of a similar activating enzyme in the hemolymph. Thus, the phenoloxidase system in Sarcophaga bullata larval hemolymph seems to be comprised of a cascade of reactions. An endogenous protease inhibitor isolated from the larvae inhibited chymotrypsin-mediated prophenoloxidase activation but failed to inhibit the cuticular activating enzyme-catalyzed activation. Based on these studies, the roles of prophenoloxidase, endogenous activating proteases, and protease inhibitor in insect immunity are discussed.     

Reexamination of phenoloxidase in larval circulating hemocytes of the silkworm, Bombyx mori

We have developed a modified method to detect phenoloxidase activity on hemocytes by using freshly prepared l-DOPA (1 mg/ml in 35% ethanol) to fix and incubate larval hemocytes. This method is more sensitive than the common method, in which hemocytes were fixed in 4% formaldehyde and then incubated with 2 mg/ml l-DOPA in water separately. Phenoloxidase assayed using this modified method can be inhibited by phenyltiourea (phenoloxidase inhibitor). After incubation with l-DOPA solution in ethanol, most prohemocytes, all plasmatocytes and young granulocytes are stained brown due to oxidation of l-DOPA into pigments, indicating that they have phenoloxidase. Oenocytoids are dimly stained because many of their cell inclusions have been released during the treatment. Large propidium-iodide-negative prohemocytes have strong phenoloxidase activity and are easily misunderstood as propidium-iodide-positive oenocytoids if the fluorescent method is not used for identification. Thus, in addition to oenocytoids and plasmatocytes, some prohemocytes and granulocytes in the silkworm also have phenoloxidase.     

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.     

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca²⁺. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.     

Differential display analysis reveals the expression of glutathione S-transferase ω and novel genes through an ITAM-containing receptor in ascidian immunocytes

The immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through antigen receptors in mammalian lymphocytes. We previously reported that an ITAM-containing receptor, ascidian hemocyte ITAM-containing receptor 1 (AhITAMR1), exists on the hemocyte surfaces of the ascidian Halocynthia roretzi, and is involved in both phagocytosis and hemocyte aggregation. In this study, we carried out differential display screening of upregulated genes during H. roretzi hemocyte aggregation and found that at least three genes are upregulated. One encodes glutathione S-transferase ω (GSTω), while the other two encode novel proteins. The expression of all three genes was induced by treatment with a specific monoclonal antibody against AhITAMR1, while their expression was inhibited by wortmannin, BAPTA-AM, and cyclosporin A. We also found that the expression of GSTω was induced by treatment with anti-T cell receptor antibody in mouse peripheral T cells. We propose that signal transduction pathways mediated by ITAM-containing receptors are conserved from ascidian hemocytes to mammalian T cells. The nucleotide sequence data reported in this study have been submitted to the DNA Data Bank of Japan (DDBJ) with accession numbers AB187220 for 18A-1, AB187221 for 20A, and AB187222 for 20G-1.     

Hemocyte-mediated phagocytosis and melanization in the mosquito<Emphasis Type="Italic"> Armigeres subalbatus</Emphasis> following immune challenge by bacteria

Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(–) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.This work was supported by NIH grant AI19769 to B.M.C. and NIH grant F31 AI50252 to J.F.H.     

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription

Summary An electrophoretic mobility variant of phenoloxidase in a lz stock of Drosophila melanogaster was identified as the A₃ component of the phenoloxidase complex by using two different activators to study enzyme activity — natural activator isolated from pupae and 50% 2-propanol. The structural gene for the A₃ proenzyme, Dox-3, was not associated with lz on the X chromosome; it mapped to the right of rdo (53.1) and left of M(2)m in the second linkage group.The lz locus affects the differentiation of the crystal cell, the type of hemocyte that carries prophenoloxidase(s) in paracrystalline form. Alleles of lz lacking paracrystalline inclusions in their hemocytes do not have phenoloxidase activity whereas alleles with paracrystalline inclusions have enzyme activity. The presence of proenzyme in the paracrystalline inclusions was demonstrated by in situ activation with natural activator or propanol followed by incubation in buffered dopa.     

Purification and properties of a collagenolytic protease produced by marine bacteriumVibrio vulnificus CYK279H

A collagenolytic enzyme, produced byVibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0 kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Asn. The optimum temperature and pH for the enzyme activity were 35°C and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8∼8.0 and 20∼35°C, respectively. The purified enzyme was strongly activated by Zn²⁺, Li²⁺, and Ca²⁺, but inhibited by Cu²⁺. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.     

Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth,Cydalima perspectalis Walker (Lepidoptera: Crambidae)

Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V_max of PO was obtained using L-dopa while the lowest K_m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC₅₀ of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K_m. Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.     

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell–cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Opsonizing properties of an isolated hemolymph agglutinin and demonstration of lectin-like recognition molecules at the surface of hemocytes fromMytilus edulis

Summary Mytilus hemolymph was found to contain an agglutinin which could be inhibited by mucin. The agglutinin was isolated by affinity chromatography using neuraminidase-treated mucin/Sepharose.In vitro phagocytosis experiments revealed that only about 5% of washed hemocytes phagocytosed yeast cells suspended in a Tris-buffered NaCl-solution, whereas yeast suspended in hemolymph was normally ingested by more than 50% of the hemocytes. This relatively high phagocytic activity was shown to depend on the presence of two serum factors: When purified agglutinin was added to saline-suspended yeast, phagocytosis rates returned to normal, demonstrating opsonizing properties of the purified agglutinin. — On the other hand, addition of Ca⁺⁺-ions to saline caused an increase of the phagocytic activity of hemocytes. This was interpreted to indicate the activation of divalent cation-dependent recognition molecules at the hemocyte surface. The function of these postulated recognition factors was demonstrated by phagocytosis inhibition tests. Their location at the hemocyte membrane became evident by binding of specific antiagglutinin IgG purified by help of an agglutinin/Sepharose column from an antiserum raised againstMytilus serum proteins. Consequently, humoral as well as cell bound agglutinin molecules are involved in the attachment of yeast cells toMytilus hemocytes which subsequently internalize foreign cells.Abbreviations DAB dimethylamino benzaldehyde - PO peroxidase - IgG immunoglobulin G     

Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris–HCl pH 7.0, 10 mM MgCl₂, 1 mM dithiothreitol at 37 °C. XorKII was easily inactivated by heating at 65 °C for 5 min, but retained most of the original activity after incubation at 37 °C for 24 h.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Purification and partial characterization ofd-(−)-lactate dehydrogenase fromLactobacillus helveticus CNRZ 32

Summary d-(–)-Lactate dehydrogenase (LDH) was purified to homogeneity from a cell-free extract ofLactobacillus helveticus CNRZ 32. The native enzyme was determined to have a molecular weight of 152 000 and consisted of four identical subunits of 38 000. This enzyme was NAD dependent fructose 1,6-diphosphate (FDP) and ATP independent. It was most active on pyruvate followed by -hydroxypyruvate as substrates. TheK _m values for pyruvate andd-(–)-lactate were 0.64 and 68.42 mM respectively, indicating that the enzyme has a higher affinity for pyruvate. The enzyme activity was completely inhibited byp-chloromercuribenzoate (1 mM) and partially by iodoacetate, suggesting the involvement of the sulfhydryl group (-SH) in catalysis. Optima for activity by the purified enzyme were pH 4.0 and 50–60°C. Limited inhibition ofd-(–)-LDH was observed with several divalent cations. Additionally, HgCl₂ was observed to strongly inhibit enzyme activity. The purified enzyme was not affected by dithiothreitol or any of the metal chelating agents examined.