Insoluble Enzyme Research Articles

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.

Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes

The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the Solubilization of 14–25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2-diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine.

An extraordinary accumulation of (−)-pinoresinol in cell-free extracts of Forsythia intermedia: evidence for enantiospecific reduction of (+)-pinoresinol

Stereoselective and enantiospecific transformation mechanisms in lignan biogenesis are only now yielding to scientific inquiry: it has been shown that soluble cell-free preparations from Forsythia intermedia catalyse the formation of the enantiomerically pure lignan, (−)-secoisolariciresinol, when incubated with coniferyl alcohol in the presence of NAD(P)H and H 2O 2. Surprisingly, (−)-pinoresinol also accumulates in this soluble cell-free assay mixture in > 96 % enantiomeric excess, even though it is not the naturally occurring antipode present in Forsythia sp. But these soluble cell-free preparations do not engender stereoselective coupling; instead, racemic pinoresinols are first formed, catalysed by an H 2O 2-dependent peroxidase reaction. An enantiospecific NAD(P)H reductase then converts (+)-pinoresinol, and not the (−)-antipode, into (−)-secoisolariciresinol. Stereoselective syntheis of (+)-pinoresinol from E-coniferyl alcohol is, however, catalysed by an insoluble enzyme preparation in F. suspensa, obtained following removal of readily soluble and ionically bound enzymes; no exogenously supplied cofactors were required other than oxygen, although the reaction was stimulated by NAD-malate addition. Thus, the overall biochemical pathway to enantiomerically pure (−)-secoisolariciresinol has been delineated.

Role of insoluble enzymes in anaerobic wastewater treatment and enzyme‐bioactivator interactions

Abstract Five insoluble hydrolases (cellulase, phosphatase, protease, lipase and urease) were localized in the solid phase of an anaerobic residual sludge. Several washing treatments were tested to evaluate the binding forces between the enzymes and the solid matter. This interaction was compared to that obtained with a bioactivator composed of mineral microparticles. Urease was found to be strongly bound and unaffected by all the washing procedures tested, whereas, the phosphatase was rapidly and completely bound but could be easily removed by a non ionic detergent. The other enzymes had intermediate binding characteristics. Attempts to degrade cellulose under batch anaerobic conditions showed an increase of the hyrolysis and acetogenesis rates due to enzyme‐activator complex.

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: Application in the semisynthesis of molecular variants of α-globin

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the α-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37°C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the α-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of α-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected α-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of α-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated α-globin. The complementary fragments from two natural mutant forms of α-globin, viz., α 1–30 (K16E) from Hb I and α 31–141 (D47H) from Hb Sealy, are readily ligated to form the double mutant α 1–141 (K16E;D47H).

Fructose 1,6-Bisphosphatase in the Green Alga Selenastrum minutum

Two isoforms of fructose 1,6-bisphosphatase are present in the green alga Selenastrum minutum. The isoenzymes can be separated with ionexchange chromatography or acid precipitation. The stability of the two isoenzymes differ largely. The acid insoluble enzyme exhibits properties similar to that of the enzyme from the chloroplasts of higher plants, i.e. an alkaline pH optima in the absence of reductant, a lower affinity for substrate, strong inhibition by phosphate, and a low sensitivity to fructose-2,6-bisphosphate and AMP. The more abundant form of the enzyme exhibits several properties indicative of heterotrophic fructose 1,6 bisphosphatases, i.e. a high affinity for substrate and sensitivity toward fructose-2,6-bisphosphate and AMP. but is absolutely dependent on a reductant for stability and activity. Evidence is provided indicating that previously reported purification protocols cause inactivation of one of the isoenzymes which could lead to the erroneous conclusion that algae have a single fructose 1,6-bisphosphatase isoenzyme.

Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate

In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymic activities involved in the oothecal sclerotization of the praying mantid, Tenodera aridifolia sinensis saussure

The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis, two soluble enzyme fractions of different activities were obtained. One fraction acted on N-acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica, also had these two activities.

DNase activity ofMicrococcal endonuclease insolubilized on corn cob

The endonuclease from S. aureus has been immobilized on ground maize cob, previously activated with tosyl chloride. Pretreatment of the support on acid before tosylation yielded the best insoluble enzyme derivatives. The catalytic activity has been evaluated as percent of total hydrolysis attained in a batch reactor using DNA as a model substrate. The derivatives prepared are very resistant to high temperatures under conditions of catalysis (24 h at 45 degrees C). For these long reaction times, the extent of hydrolysis in the presence of small amounts of organic solvent (dimethyl sulfoxide at 2 percent) is larger than in plain buffer (Tris). This type of derivative could be very useful for the removal of nucleic acids from single-cell protein concentrates.

Natural disturbance and the activity of Trichoderma viride cellulase complexes

The stability of organic matter-cellulase complexes was investigated using the enzymes of Trichoderma viride as a model. Soluble enzyme complexes were produced by reaction of cellulase with water-extractable substances from deciduous and pine litters. Insoluble enzyme complexes were produced by adsorption of cellulase to litter particles. Freeze-thaw and wet-dry events were used to evaluate the stability of the cellulase complexes. Soluble enzyme complexes generally sustained higher activity to repeated freeze-thaw events than uncomplexed enzymes. After 10 events, uncomplexed enzymes retained 42–56% of their initial activity while soluble enzyme complexes retained 60–145%. There were no consistent differences in response among enzyme types or extract sources. Among insoluble enzyme complexes, only β-glucosidase consistently displayed enhanced activity relative to free enzyme. Wet-dry events affected activity more severely than freeze-thaw ones. The stability of adsorbed cellulase varied among enzymes with β-glucosidase retaining 55–105% of its initial activity after 10 events, exocellulase retaining 12–60% after 10 events, and endocellulase 0–5%. The stability patterns of the enzyme complexes are suggested as being the result of two confounded processes: enzyme inactivation and complex dissociation.

Mixed enzymic reaction--internal diffusion kinetics of nonuniformly distributed immobilized enzymes. The system agarose-micrococcal endonuclease.

Two types of (CNBr-activated) Agarose-staphylococcal endonuclease derivatives have been prepared, one with the enzyme uniformly distributed in the support, and the other with the enzyme preferentially bound in the most external part of the support particles; the latter were obtained using agarose of very small pores and a high degree of activation. Quantitative enzyme distribution has been determined by scanning fluorescence microscopy. With these insoluble enzyme derivatives, a kinetic study for the hydrolysis of a mononucleotide has been carried out. A simple theoretical model for nonuniformly distributed insoluble enzyme derivatives, which considers only the case of mixed enzymic reaction-internal diffusion kinetics, is proposed. The experimental data agree very well with the predictions of the model.

Formula omitted]: Biosynthesis in insect cuticle and possible role in sclerotization

N-β- alanylnorepinephrine (NBANE), a metabolite of N-β- alanyldopamine (NBAD) in pupal cuticle of the tobacco hornworm, Manduca sexta, was identified by analysis of hydrolytic products, by comparison of HPLC retention times with a synthetic standard and by fast atom bombardment mass spectrometry. NBANE and NBAD were abundant in the brown cuticle of M. sexta pupae, Periplaneta americana adults, Tribolium castaneum adults and Musca domestica puparia. Side chain modification of NBAD was rapid during the first few hours of sclerotization as evidenced by a dramatic increase in NBANE extracted from M. sexta pupal cuticle with cold 1.2 M HCl. In later stages of sclerotization less NBANE was recovered indicating further metabolism and possible involvement in the formation of crosslinks to cuticular components. Although similar amounts of the two catecholamines were extracted by 80% methanol from pupal cuticle which was just starting to tan, over 15-fold more NBANE than NBAD was recovered with cold dilute HCl. Low pH may be necessary to remove physically entrapped NBANE from the insoluble polymeric matrix of the cuticle, or NBANE released by the acid treatment may be a hydrolytic product from NBAD covalently linked by its β-carbon to cuticular components. NBANE was synthesized from NBAD by an insoluble enzyme in tanning pupal cuticle that also catalyzed to a lesser extent the β-hydroxylation of N- acetyldopamine (NADA). However, NADA was a better precursor than NBAD for compounds that yield 3,4-dihydroxphenylketoethanol after heating in 1 M HCl. Therefore, two pathways were observed in tanning cuticle for side chain metabolism of the N- acylated dopamines, and the type of acyl group influenced the relative amounts of different sclerotizing agents produced.

Lipase catalyzed reactions and strategy for alcohol resolution

Asymmetric hydrolysis, ester interchange and ester formation were carried out for menthol resolution with Candida cylindracea lipase. The reactions were realized under emulsified conditions with water soluble enzyme and in organic medium with an insoluble enzyme preparation. In the latter case, an enhancement of the enantioselectivity uas observed. The potentialities of ester interchange and ester formation for preparative alcohol resolutions are discussed.

Characterization of glucuronosyl transferase from Mucor rouxii: Requirement for polyuronide acceptors

Most of the glucuronosyl transferase activity present in cell-free extracts from Mucor rouxii was found associated with a mixed membrane fraction (MMF). Treatment of this fraction with neutral detergents did not extract any transferase activity, but the insoluble enzyme became dependent on the addition of an exogenous primer, mucoric acid being more efficient than mucoran. Both polyuronides acted as true acceptors of glucuronosyl residues and were not merely enzymatic activators. Incubation of Triton-extracted MMF with UDP-glucuronic acid in the presence of UDP-GlcNAc, UDP-Glc, and UDP-Gal brought about a stimulation in the incorporation of glucuronic acid. This result was taken as evidence that, under these conditions, a heteropolymer was synthesized. Treatment with neutral detergents of native membrane fractions previously incubated with radioactive substrate extracted most of the incorporated radioactivity in the form of a polymer containing both protein and uronic acids. Treatment of this polymer with Pronase digested the protein, and radioactivity appeared in the form of low-molecular-weight compounds. Some properties of the glucuronosyl transferase treated with detergents were as follows: optimum temperature for enzyme activity was 26°C; the enzyme required a divalent metal ion, 4 m M MnCl 2 being the most efficient; and the enzyme showed biphasic kinetics with a Michaelis constant of 0.73 m M at low substrate concentrations.

Immobilization of cane invertase on bentonite

Sugar-cane invertase (β- d-fructofuranoside fructohydrolase, EC 3.2.1.26) immobilized on bentonite clay in 0.05 m acetate buffer, pH 4.5, has been shown to be capable of hydrolysing sucrose. The bentonite-invertase (BI) complex gave 55.5% retention of enzyme activity on the surface. A further 17 and 22% increase in retention of enzyme activity was obtained using the covalent linking agents, cyanuric chloride and thionyl chloride, giving bentonite-cyanuric chloride-invertase (BCCI) and bentonite-thionyl chloride-invertase (BTCI) complexes. Concentrations of acetate buffer >0.2 M disrupt the bentonite-invertase complexes. The immobilized invertase complexes showed high temperature optima (60–65°C) and high thermal stability compared to the free enzyme. The pH profiles of the free and immobilized enzyme were the same. The rate of hydrolysis of sucrose was increased using immobilized enzymes, which required a higher substrate concentration than the free enzyme. The insoluble enzyme conjugate-carrier complexes when used for sucrose hydrolysis in a batch process showed 53.1 (BI), 57.4 (BCCI) and 59.6% (BTCI) conversions, respectively, in 12 h, compared to 42.3% conversion in 24 h with the free enzyme. The immobilized invertase complexes can be used for sucrose inversion for about five cycles. The application of this immobilization procedure may help in the removal of invertase from cane juice to reduce sugar losses in industry.

Characterization of phenol oxidase in a pseudophyllidean cestode Penetrocephalus ganapatii

The biochemical characteristics of phenol oxidase obtained from Penetrocephalus ganapatii have been investigated using manometric and electrophoretic techniques. Phenol oxidase was found to have maximum activity at pH 7·4, the Michaelis constant for dopamine was 189 μM and the enzyme was stable between 20 and 40°C. Potassium cyanide, sodium diethyldithiocarbamate and phenylthiourea strongly suppressed enzyme activity. The Ki values for potassium cyanide, diethyldithiocarbamate and phenylthiourea were 33μM, 700 μM and 938 μM respectively. The properties of soluble and insoluble enzymes to various phenolic substrates are discussed.

Effect of immobilization on stability and kinetic properties of α‐L‐fucosidase from Turbo cornutus

An amount of alpha-L-fucosidase from T. cornutus liver was copolymerized with glutaraldehyde using bovine serum albumin as the carrier protein. The properties of the native, the soluble enzyme polymer complex, and the insoluble enzyme polymer complex were studied and compared under various conditions of pH, temperature, substrate, and inhibitor concentration. Native alpha-L-fucosidase was heat labile and lost more than 85% of its activity when incubated at 55 degrees C for 5 min. In contrast, under equivalent incubation conditions, both the soluble and the insoluble enzyme polymer complexes exhibited enhanced resistance to thermal inactivation and after 5 min lost only 65 and 40% of their original activity, respectively. Polymerzation also resulted in the shift of pH optima towards the acidic range, a decrease in activation energy and a change in the apparent K(m) values towards the p-nitrophenyl-alpha-L-fucopyranoside substrate.

Partial purification and characterization of neutral proteases in lymph nodes of rats with experimental allergic encephalomyelitis.

Two kinds of neutral protease activities in lymph nodes from Lewis rats with acute experimental allergic encephalomyelits (EAE) have been separated and partially purified and characterized. A soluble enzyme preparation enriched by gel filtration and ion-exchange chromatography hydrolyzes myelin basic protein, polylysine, and other basic proteins with an optimum pH at 6.0-6.5. It is inhibited by p-chloromercuribenzoate, and thus appears to be a mixture of thiol proteases. Another fraction containing proteolytic enzyme activity is strongly bound to the insoluble lymph node residue, and it also hydrolyzes myelin basic protein and histone, but not polylysine. It has a pH optimum above 7.5, is inhibited by phenylmethylsulfonyl fluoride, thus resembling elastase, but does not hydrolyze elastin-Congo red. The insoluble enzyme preparation hydrolyzes basic protein to 4-5 peptides in a pattern on polyacrylamide gels resembling that of the hydrolysis of basis protein by whole lymphocytes; the soluble enzyme mixture produces small fragments not retained on gels. Lymphocytes are a major component of the cells infiltrating the nervous system in experimental allergic encephalomyelitis, and neutral proteases contained in these cells may contribute to the degradation of myelin, especially of the basic protein.

A novel preparation of human platelet lipoxygenase: Characteristics and inhibiton by a variety of phenyl hydrazones and comparisons with other lipoxygenases

Acetone-pentane powder preparations of human blood platelets were prepared and the characteristics of the 12L-lipoxygenase were studied using measurements of oxygen consumption. No sharp pH optimum with equal reaction velocities was observed over a range of pH 7.5–8.5 in a variety of buffers. The enzyme could be easily solubilized in 1% deoxycholate, and in this form was moderately stable to heat. Of 10 divalent cations tested at a concentration of 3.7 · 10 −3 M, only zinc and tin were inhibitory. None of the other ions was stimulatory. The products of the oxidation of arachidonate were characterized from both soluble and insoluble enzyme preparations. With the insoluble suspension, 55% of the added arachidonate was recovered as 12-hydroxy-5,8,10, 14-eicosatetraenoic acid (12-HETE) and 27% appeared as trihydroxyeicosatetraenoic acid isomers (THETEs). With the soluble preparation, 84% of the arachidonate appeared as 12-HETE and only 5% as THETEs. The Michaelis constants for dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid substrates are presented. A series of phenyl hydrazone inhibitors of various structural types were discovered to be potent inhibitors of the human platelet lipoxygenase. The sensitivities of this enzyme to these inhibitors were compared to soybean lipoxygenase and sheep seminal vesicular cyclodioxy genase. In general, the soybean enzyme was most sensitive; the sheep seminal vesicular cyclodioxygenase was the least sensitive and the human platelet lipoxygenase was intermediate between the two. The K i values for two phenyl hydrazone inhibitors with both soybean and human platelet lipoxygenases is presented.

Human Spleen Insoluble Fibrinolytic Proteinase Acting at Neutral pH: Its Partial Purification and Characterization

An insoluble fibrinolytic enzyme with a molecular weight of approximately 30,000, was purified from the human spleen. A single protein band possessing fibrinolytic activity was obtained on polyacrylamide gel disk electrophoresis at pH 4.5. The enzyme, tentatively termed spleen fibrinolytic proteinase (SFP), degraded fibrinogen at neutral pH following Michaelis-Menten kinetics. The fibrinogenolytic activity was not inhibited by t-AMCHA, a specific plasmin inhibitor. SFP barely degraded certain synthetic ester or polypeptide substrates for trypsin, chymotrypsin, plasma, Xa, elastase and collagenase. These results indicate a different nature for SFP compared to other enzymes examined. SFP was found to digest no elastin and its fibrinogenolytic activity was strongly inhibited by STI, indicating that it was not an elastase. SFP required neither Zn++ nor CA++ for its fibrinogenolytic activity, indicating that it differed from metal-dependent proteinases such as collagenase. SFP was inhibited by DFP but not by TLCK, suggesting that it contains an active serine residue, but no trypsin type histidine at its active center. These results appear to show that SFP is a unique proteinase in the spleen, which is capable of degrading fibrin and fibrinogen at neutral pH.