Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Identification of Ionizable Groups Essential to the Activity of Isomalto-dextranase from Arthrobacter globiformis

The active site of isomalto-dextranase from Arthrobacter globiformis was investigated by kinetic and chemical-modification methods. The ionization constants, pK_e1 and pK_e2, of the essential ionizable groups 1 and 2 of the free enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The pK_el and pK_e2 both shifted to higher pH when the dielectric constant of the reaction mixture decreased. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate. Modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, modifying carboxyl residues specifically, resulted in inactivation of the enzyme, and isomaltotriose protected the enzyme against such inactivation. These findings also indicated that the carboxyl groups were essential to the enzyme activity.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

Purification and characterization of halohydrin hydrogen-halide lyase from a recombinant Escherichia coli containing the gene from a Corynebacterium sp.

Summary An enzyme catalyzing the interconversion of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) was purified from Escherichia coli JM109/ pST001, which carried the gene from Corynebacterium sp. N-1074. The enzyme was crystallized by the addition of ammonium sulphate. The enzyme had a relative molecular mass (M_r) of about 105 000 and consisted of four subunits identical in M_r (approx. 28 000). The enzyme catalysed both the transformation of various halohydrins into the corresponding epoxides with liberation of halide and its reverse reaction. These facts indicated that the enzyme was halohydrin hydrogen-halidelyase. Offprint requests to: T. Nagasawa     

Isolation,Purification, and Properties of Lytic Enzyme from Polysphondylium pallidum Myxamoebae

Two lytic enzymes (enzyme I and enzyme II) that lysed Micrococcus lysodeikticus were isolated from the crude extract of Polysphondylium pallidum myxamoebae grown in the presence of Klebsiella aerogenes by precipitation with protamine sulfate and by chromatography on DEAE-Sepharose CL-6B. Enzyme I was further purified by gel filtration on a Superose12 column, and enzyme II by chromatography on a MonoQ HR 5/5 column and gel filtration on a Superose12 column. Enzyme I was a basic protein, while enzyme II was acidic. The molecular weights of enzyme I and II were about 14,000 and 22,000, respectively by SDS-polyacrylamide gel electrophoresis. Optimum pHs for the activity were 5.0 for enzyme I and between 3.5 and 4.0 for enzyme II. The maximum activity of enzyme I and II was obtained at 65°C and 45°C to 55°C and at ionic strength of 0.0075 to 0.03 and 0.06, respectively. Both enzymes cleaved the glycosidic bond of β(1,4)-N-acetylmuramyl-acetylglucosamine of the cell wall peptidoglycan of Micrococcus lysodeikticus. These results indicate that the two lytic enzymes of Polysphondylium pallidum myxamoebae are N-acetylmuramidases.     

Pencillin amidase production by Bacillus megaterium

Penicillin amidase, an enzyme which hydrolyzes benzylpenicillin to 6-aminopenicillanic acid and phenylacetic acid, is produced by Bacillus megaterium ATCC 14945 as an extracellular enzyme. We used this system as a model to examine the effects of nitrogen, sulfur, and phosphorous limitation on enzyme production in continuous culture. For these studies, we developed a minimal medium for B. megaterium which contained histidine as the sole nitrogen source. Batch experiments showed that this enzyme is produced as a growth-associated metabolite. Enzyme production was shown to be a function of the growth-limiting conditions and the concentration of the inducer, phenylacetic acid. Sulfur limitation in continuous culture yielded enzyme activities approximately three to five times those observed in nitrogen- and phosphorous-limited chemostats. These results are discussed in terms of the environment's influence on enzyme production in continuous culture.     

Properties of partially purified leaf ornithine aminotransferase of Brassica juncea under salt stress

Properties of the partially purified L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) of leaves of Brassica juncea salt tolerant somaclone SR3P6-2 and its parent cv. Prakash were studied. The enzyme from the somaclone SR3P6-2 was relatively more efficient in terms of its Km, Vmax, and Ea (activation energy) and required higher levels of chlorides for inhibition as compared to the enzyme from the parent cv. Prakash. These results suggest some salt-stress related changes in the enzyme.     

Recycle of cellulases and the use of lignocellulosic residue for enzyme production after hydrolysis of steam-pretreated aspenwood

Summary Various modes of substrate and enzyme addition were used to hydrolyze a 10% concentration (w/v) of steam-exploded, water-and-alkali extracted aspenwood withTrichoderma harzianum E58 cellulases. Although cellulose conversion was high (94–100%), enzyme recovery was low in all cases. Low enzyme recovery was due to a combination of thermal inactivation and adsorption of the cellulases onto the lignocellulosic residue. Enzyme recycle was not feasible as the activity of the recovered cellulases towards crystalline cellulose was low. However, the residual material from enzyme hydrolysis was a suitable carbon source for cellulase enzyme production byT. harzianum based on enzyme yield and hydrolytic potential. These residues could only be used up to a 1% substrate concentration, since at higher substrate loadings cellulase production was reduced, likely because of lignin inhibitors.     

Calcium Ions and a Secreted Peroxidase in <Emphasis Type="Italic">Euphorbia characias</Emphasis> Latex are Made for Each Other

This minireview deals of a protein, a class III secreted peroxidase, present as unique isoform in the latex of the perennial Mediterranean shrub Euphorbia characias. The paper reports on the molecular properties, on the structures (primary, secondary and tertiary), and on the catalytic mechanism of this enzyme. Here is also reported the extraordinary effect of calcium ions on the structure and on the enzyme activity of Euphorbia peroxidase. These ions can either enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to execute different metabolic pathways toward the same substrate. This review will give a valuable reference to the peroxidase fans and the general readers will find many thorough suggestions for future researches giving birth to new studies and important discoveries.     

Über Vorkommen und Lokalisation eines flavonolumwandelnden Enzyms in Pflanzen

Summary The occurrence and distribution of an enzyme converting flavonols to 2,3-dihydroxy flavanones has been measured in various plants and found to occur in all plants tested.In garbanzo bean, Cicer arietinum L., the enzyme is found mainly in roots, hypocotyls, epicotyls and cytoledons while the other organs, rich in flavonols, possess much lower levels of the enzyme. In garbanzo seedlings the enzyme is formed between the second and sixth day after germination and appears parallel to the phenylalanine ammonia-lyase. The data indicate that the enzymes for both flavonol biosynthesis and turnover are formed simultaneously. These results further support earlier observations that regulation of flavonoid formation and regulation of turnover seem to be dependent on each other.The activity of the flavonol-converting enzyme does not increase in Cicer arietinum plants transferred to darkness though under such conditions flavonol turnover is accelerated.In Pisum sativum, Glycine max and Sinapis alba the flavonol converting enzyme is more evenly distributed over all organs, so that a correlation between flavonol content and enzyme is less obvious.The data are discussed with respect to intracellular regulation of flavonol turnover.     

Proteolysis of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the K _m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.     

Localization of functional β-xylosidases,encoded by the same single gene,xlsIV (xlnD), from Aspergillus niger E-1

Cell wall-associated β-xylosidase was isolated from Aspergillus niger E-1 and identified as XlsIV, corresponding to the extracellular enzyme XlnD reported previously. xlsIV was transcribed only in the early cultivation period. Cell wall-associated enzyme activity gradually decreased, but extracellular activity increased as the strain grew. These results indicate that XlsIV (XlnD) was secreted into culture after localizing at cell wall.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

THE REGIONAL DISTRIBUTION OF CYTIDINE 5''-MONOPHOSPHO-N-ACETYL-NEURAMINIC ACID SYNTHETASE IN CALF BRAIN

—The enzyme cytidine 5′-monophospho-N-acetylneuraminic acid synthetase was studied in different parts of the calf brain. Characterization of partial purified enzyme preparations from cortical grey matter and corpus callosum by means of pH optima and K_m values, showed the enzyme of grey and white brain areas to be identical. Unexpectedly the regional differences of the enzyme activities per g wet tissue and per mg protein were very slight. From the presence of the enzyme in pure white brain areas, which are known to be poor in neuronal perikarya, and the fact that the enzyme is localized in the cell nucleus, we concluded that cytidine 5′-monophospho-N-acetylneuraminic acid is produced in glia cell nuclei and that it is very likely that biosynthesis of sialo-glycoproteins and/or ganglio-sides occurs within glia cells. The enzyme activity per μmol DNA-P is somewhat higher in grey than in white regions, indicating a slightly higher activity per neuronal than per glial nucleus. The regional differences of lipid and protein-bound sialic acid and RNA show a striking similarity and contrast to those of the enzyme. These differences are interpreted in terms of a differential content in neurons and glia cells.     

Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants. Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)     

Allocation of random amplified polymorphic DNA markers and enzyme activities toAspergillus nidulans andAspergillus tetrazonus chromosomes

Chromosome-substituted haploid segregants of anA. nidulans × A. tetrazonus somatic hybrid were used to allocate several random amplified polymorphic DNA and isoenzyme markers to parental chromosomes. Twenty-six amplified DNA fragments, and nine isoenzyme activities, including lactate dehydrogenase, superoxide dismutase, and arylesterase isoenzymes were assigned to chromosomes. Chromosome-specific markers were found for eachA. nidulans andA. tetrazonus chromosome. These markers could be used to saturate the genetic map ofA. nidulans. The formation of two secondary metabolites was also assigned to chromosomes III and VIII. Attempts were made to allocate extracellular enzyme activities to parental chromosomes, mostly without success, possibly because multiple enzyme forms located on different chromosomes could be responsible for the production of an enzyme activity.     

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHΔCBM and EngYΔCBM devoid of the CBM lost activity toward all substrates including CMC. EngKΔCBM and EngMΔCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty-nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley-Liss, Inc.     

Enzymatic activity and thermal stability of PEG-α-chymotrypsin conjugates

α-Chymotrypsin was chemically modified with methoxypoly(ethylene glycol) (PEG) of different molecular weights (700, 2,000, and 5,000 Da) and the amount of polymer attached to the enzyme was varied systematically from 1 to 9 PEG molecules per enzyme molecule. Upon PEG conjugation, enzyme catalytic turnover (k _cat) decreased by 50% and substrate affinity was lowered as evidenced by an increase in the K _M from 0.05 to 0.19 mM. These effects were dependent on the amount of PEG bound to the enzyme but were independent of the PEG size. In contrast, stabilization toward thermal inactivation depended on the PEG molecular weight with conjugates with the larger PEGs being more stable.     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.