Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase of Clostridium thermocellum

The crude extracellular cellulase from Clostridium thermocellum was oxidatively inactivated by air and inhibited by sulfhydryl reagents. Activity-loss was prevented and reversed by the addition of a high concentration (10 mM) dithiothreitol (DDT) at zero time and up to 24 h respectively. In the presence of a low concentration (0.4 mM) of DTT, the enzyme was more rapidly inactivated than in air alone. This was probably due to autoxidation of the low DTT concentration to H₂O₂ as shown by its prevention by a high DTT concentration, exclusion of air, or catalase; and by the oxidative inactivation of the enzyme by H₂O₂. The inactivation by H₂O₂ could be prevented by a high concentration of DTT but not by air exclusion. EDTA protected the enzyme from inactivation in air by a low concentration of DTT or by H₂O₂. This is presumably due to the role of metals in oxidation of SH groups. Furthermore, copper (5 M) also caused inactivation and this was prevented by the presence of a high DTT concentration. Even in the protective atmosphere of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents such as o-phenanthroline and -¹-dipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture of Fe²⁺ and Fe³⁺. These data suggest that the clostridial cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The endo--glucanase component of the cellulase complex was not susceptible to oxidative inactivation.Abbreviations DTT dithiothreitol - CMC carboxymethylcellulose - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - p-CMB p-chloromercuribenzoic acid     

Major differences in isoforms of starch-branching enzyme between developing embryos of round- and wrinkled-seeded peas (Pisum sativum L.)

In order to determine whether round-and wrinkled-seeded peas (Pisum sativum L.) differ in the activity and properties of starch-branching enzyme (1,4--D-glucan, 1,4--D-glucan-6-glycosyl transferase; EC 2.4.1.18) in their developing embryos, essentially isogenic lines of peas, differing only at the r (rugosus) locus that confers the round (RR, Rr) or wrinkled (rr) phenotype, were studied. Activity of the enzyme rises rapidly from an early stage of development in embryos of round peas, but only at later stages in embryos of wrinkled peas. The purified enzyme from mature embryos of round peas can be resolved into two isoforms that differ in molecular weight and in their ability to branch amylose. The purified enzyme from mature embryos of wrinkled peas is a single protein with the same molecular weight and branching properties as one of the isoforms from embryos of round peas. The difference in activity of starch-branching enzyme between embryos of round and wrinkled peas is likely to be due to the absence from embryos of wrinkled peas of one of the isoforms occurring in embryos of round peas.Abbreviations DEAE diethylaminoethyl - FW fresh weight - kDa kilodalton - SDS sodium dodecyl sulfate     

Cloning, sequencing and expression of a gene encoding a 73 kDa xylanase enzyme from the rumen anaerobe Butyrivibrio fibrisolvens H17c

Summary The cloning, expression and nucleotide sequence of a 3 kb DNA segment on pLS206 containing a xylanase gene (xynB) from Butyrivibrio fibrisolvens H17c was investigated. The open reading frame (ORF) of 1905 by encoded a xylanase of 635 amino acid residues (M_r 73156). At least 850 by at the 3 end of the gene could be deleted without loss of xylanase activity. The deduced amino acid sequence was confirmed by purifying the enzyme and subjecting it to N-terminal amino acid sequence analysis. In Escherichia coli C600 (pLS206) cells the xylanase was localized in the cytoplasm. Its optimum pH for activity was between pH 5.4 and 6, and optimum temperature 55° C. The primary structure of the xylanase showed a significant level of identity with a cellobiohydrolase/endoglucanase of Caldocellum saccharolyticum, as well as with the xylanases of the alkaliphilic Bacillus sp. strain C-125, B. fibrisolvens strain 49, and Pseudomonas fluorescens subsp. cellulosa.Abbreviations ORF open reading frame - pNPCase p-nitrophen-yl--d-cellobiosidase - (xynB) gene coding for XynB - XynB xylanase     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

Circadian rhythms in crassulacean acid metabolism: phase relationships between gas exchange,leaf water relations and malate metabolism in Kalanchoë daigremontiana

Hypocotyls of 5-d-old etiolated soybean seedlings (Glycine max (L.) Merr. cv. Altona) were treated with (a) dithiothreitol (DTT) or one of the sulfhydryl-binding reagents N-ethylmaleimide (NEM), p-hydroxymercuribenzoate (PMB) und p-chloromercuribenzene sulfonic acid (PMBS), (b) one of the sulfhydryl reagents in combination with DTT, (c) sulfhydryl reagent subsequent to treatment with DTT, and (d) PMBS followed by DTT. Glyceollin was extracted 24 and 48 h after initiation of treatment. The order of decreasing glyceollin-eliciting activity was PMBSDTT>PMBNEM. Elicitor effectiveness of sulfhydryl reagents and their reactivity with either L-cysteine or sulfhydryl groups in soybean hypocotyls were not strictly correlated. Mixtures of sulfhydryl reagent and DTT, pretreatment of hypocotyls with DTT and subsequent application of either PMB or PMBS, as well as application of PMBS prior to DTT induced less glyceollin than sulfhydryl reagents alone. In contrast, such pretreatment did not appreciably alter glyceollin accumulation elicited by NEM. The results indicate that glyceollin synthesis can be regulated by interaction with sulfhydryl groups located mainly at the outer surface of the plasmalemma.Abbreviations DTT DL-dithiothreitol - NEM N-ethylmaleimide - PMB p-hydroxymercuribenzoate (sodium salt) - PMBS p-chloromercuribenzene sulfonic acid     

Assimilatory nitrate reductase from Acinetobacter calcoaceticus

A soluble nitrate reductase from the bacterium Acinetobacter calcoaceticus grown on nitrate has been characterized. The reduction of nitrate to nitrite is mediated by an enzyme of 96000 molecular weight that can use as electron donors either viologen dyes chemically reduced with dithionite or enzymatically reduced with NAD(P)H, through specific diaphorases which utilize viologens as electron acceptors. Nitrate reductase activity is molybdenum-dependent as shown by tungstate antagonistic experiments and is sensitive to -SH reagents and metal chelators such as KCN.The enzyme synthesis is repressed by ammonia. Moreover, nitrate reductase activity undergoes a quick inactivation either by dithionite and temperature or by dithionite in the presence of small amounts of nitrate. Cyanate prevents this inactivating process and can restore the activity once the inactivation had occurred, thus suggesting that an interconversion mechanism may participate in the regulation of Acinetobacter nitrate reductase.Abbreviations EDTA ethylenediaminetetraacetate - BV benzyl viologen - MV methyl viologen - MW molecular weight - NEM N-ethylmaleimide - p-HMB p-hydroxymercuribenzoate - DCPIP 2,6-dichlorophenol-indophenol - FMN flavin mononucleotide - FAD flavin adenine dinucleotide - KCNO potassium cyanate     

Purification and properties of an extra cellular xylanase enzyme ofClostridium strain SAIV

An extracellular xylanase enzyme fraction A from a mesophilicClostridium strain SAIV was purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration and DEAE-Sephadex A-50 ion exchange. The xylanase exhibited a molecular weight of 30,000 and it was stable upto 55° C with an optimum temperature of 50° C. It was most stable between pH 5–7, with an optimum pH of around 6. The Km value was 7.0 mg·xylan ml^-1 and V_max was 36 mol·xylose liberated mg^-1 min^-1. Carboxymethyl cellulose, filter paper cellulose and 4-p-nitrophenyl -D-xylopyranoside were not hydrolysed. The specific activity of xylanase fraction A (9.8 U mg^-1) is 2–10 fold higher than the specific activity of xylanase in other mesophilic, xylanolytic, obligate anaerobic bacteria. A minor fraction of xylanase activity designated as xylanase B was also obtained supporting the view that the multiplicity of xylanases is common in microorganisms.     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Ubiquinol regeneration by plasma membrane ubiquinone reductase

Summary Several enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by high-performance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited byp-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochromeb ₅ reductase catalyzed the reduction of the natural ubiquinone homologue coenzyme Q₁₀ at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochromeb ₅ reductase in the regeneration of endogenous ubiquinol.Abbreviations AAPH 2,2-azobis-(2-amidinopropane) hydrochloride - CoQ coenzyme Q, ubiquinone - CoQH₂ reduced coenzyme Q, ubiquinol - pHMB p-hydroxymercuribenzoate     

Characterization of maltose biosynthesis from α-d-glucose-1-phosphate in Spinacia oleracea. L.

The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (K_m 1.5 mmol l^-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l^-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [³²P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru fructose - Glc glucose - -G1P -d-glucose-1-phosphate - -G1P -d-glucose-1-phosphate - G6P d-glucose-6-phosphate This enzyme is tentatively called maltose synthase in this publication     

Isolation and Characterization of Hieronymain II, Another Peptidase Isolated from Fruits of Bromelia hieronymi Mez (Bromeliaceae)

From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5–9.0 on casein and at pH 7.3–8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide ( / ). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^–1 min^–1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with subunits complex, the enzyme activity completely disappeared, whereas when subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Cryptopolyploidy inBunias (Brassicaceae) revisited — A flow-cytometric and densitometric study

The concern of the present analysis is the hypothetical cryptopolyploidy, a concept basically of historical interest only, but discussed again by Battaglia (1996) in a recent treatment of the term and its historical background. Melinossi (1935), while reanalyzing erratic observations on the crucifersBunias erucago andB. orientalis by Jaretzky (1928a), found 2n = 14 in both species but twice the chromosome volume inB. erucago compared withB. orientalis. Melinossi considered cryptopolyploidy inB. erucago, i.e., she discussed pairwise fused chromosomes on a tetraploid basis or endoreduplicated (and thus binemic) chromosomes in this species. Cryptopolyploidy has also been claimed by Pannocchia-Laj (1938) inVinca difformis (Vincaceae). Battaglia (1996) criticized the term cryptopolyploidy because, in his opinion, the genuinely polyploid status of these plants is not hidden (crypto) but phenotypically (from herbarium specimens) recognizable. He coins the term phenopolyploidy, i.e., phenotypic polyploidy disagreeing with the karyotype numerically evalated. We measured genome size ofB. orientalis andB. erucago (both 2n = 14) by Feulgen densitometry and propidium iodide flow cytometry. Surprisingly,B. erucago (the annual species with 2.13 pg, 1 C) turned out to have only 0.81-fold the DNA amount ofB. orientalis (the perennial species with 2.64 pg, 1 C). Therefore, any kind of genetically polyploid status inB. erucago is out of the question. Only speculative significance can be ascribed to the terms cryptopolyploidy and phenopolyploidy.     

Influence of reagents reacting with metal, thiol and amino sites of catalytic activity and l-phenylalanine inhibition of rat intestinal alkaline phosphatase

1. Studies on the inactivation of rat intestinal alkaline phosphatase by several metal-binding agents, namely EDTA, 8-hydroxyquinoline, pyridine-2,6-dicarboxylic acid, αα′-bipyridyl, o-phenanthroline and sodium cyanide, indicated the functional role of a metal, probably zinc, in the catalysis. The metal ligands lowered stereospecific uncompetitive inhibition of the enzyme by l-phenylalanine by an extent that paralleled the decline in enzyme activity. 2. The thiol reagents p-hydroxymercuribenzoate, iodoacetamide and iodine inactivated rat intestinal phosphatase. The enzyme could be protected from inactivation by either cysteine or substrate. The l-phenylalanine inhibition remained unchanged only in the presence of moderately inactivating concentrations of the thiol reagents. 3. Inactivation of the enzyme by the amino-group-blocking reagent, O-methylisourea, provided ample evidence for the participation in the catalysis of the -amino group of lysine. At the same time, l-phenylalanine inhibition remained unaltered even when the enzyme was strongly inactivated. This -amino-group-blocked enzyme exhibited no change in migration in starch gel, in contrast with enzyme treated with acetic anhydride, formaldehyde or succinic anhydride. The Michaelis constant of the enzyme was enhanced by such modifications, but the optimum pH remained the same. 4. d-Phenylalanine acted as a competitive or `co-operative' activator for intestinal alkaline phosphatase after it had been modified by acetylation.     

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.