Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts

Formation of pectinase system in Erwinia aroideae was stimulated to a considerable extent when the cells were incubated in a pectin medium containing carrot extracts. The active factor in the extract was purified about 30 fold by ethanol precipitation, and further purification was achieved by ninhydrin treatment, charcoal adsorption, dialysis and gel filtration with Sephadex G-10. Although crude carrot extract preparation also stimulated protease formation in this organism, no stimulating activity for protease formation was found in the purified factor. Acetate and butyrate which had been shown to stimulate pectinase formation, were found to stimulated protease formation as well. Pectinase formation by this organism was also stimulated by polyamines and inorganic phosphate to a considerable extent.     

Some Properties of Transglycosylation Activity of Sesame β-Glucosidase

The transglycosylation reaction of partially purified β-glucosidase from sesame seeds with cellobiose is described. Sesame β –glucosidase was partially purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the enzyme was 200,000 by gel filtration. Sesame β-glucosidase showed strong transfer activity to synthesize the trisaccharide from cellobiose. The optimum pH and temperature of the transglycosylation reaction were pH 4.0 and 60°C.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle

Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 °C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi.     

Production and characteristics of inhibitory factor of raw starch digestion from Aspergillus niger

Summary The glucoamylase preparation of Aspergillus niger 19 inhibited the raw starch digestion by it at high enzyme concentration. The inhibitory factor (IF) was isolated from the glucoamylase preparation by heat treatment and purified by DEAE-Sephadex A-25 column chromatography, an initial Sephadex G-50 gel filtration followed by SP-Sephadex C-25 column chromatography (twice) and then second Sephadex G-50 gel filtration. The IF thus purified was homogenous in polyacrylamide gel electrophories. The inhibitory activity of IF increased with the increasing IF concentration but decreased with an increasing quantity of raw starch or enzyme concentration. The IF had no effect on the hydrolysis of boiled soluble starch. It was completely adsorbed onto raw starch. The IF had a molecular weight of about 10,500. It was abundant in hydroxy amino acids such as threonine and serine. Xylose, mannose, glucose, galactose, and galacturonic acid were present in it.     

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.     

Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose High Performance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF), and showed maximal activity at pH 10, having a molecular weight of about 33 kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl₂ but partially inhibited by CaCl₂, CuCl₂, CoCl₂, MnCl₂, and ZnCl₂, and not inhibited by ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD₅₀ values of 0.30 and 0.27 μg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy. Received: 12 April 1996 / Accepted: 31 July 1996     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Biochemical characterization of H9/25, an allospecificity encoded by the Ly-6 region

H9/25, an allospecificity encoded by the Ly-6 region, was biochemically characterized. It was sensitive to pepsin and heat treatment, but was resistant to periodate oxidation. Its apparent molecular weight was approximately 12 000 daltons by gel filtration. The antigenic molecule was partially purified by gel filtration and antibody affinity chromatography. The partially purified antigen molecule was radioiodinated, immunoprecipitated with monoclonal antibody H9/25, and analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiograph showed the molecular weight of H9/25 to be approximately 15000 daltons under reducing conditions. These results indicate that H9/25 is a protein with a single polypeptide chain of 12000–15000 daltons molecular weight, and the antigenic specificity is carried by a peptide but not a carbohydrate moiety.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

Purification and characterization of the amylase of B. subtilis NRRL B3411

The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca⁺⁺ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Isolation and Characterization of Nα-Benzyloxycarbonyl Amino Acid Urethane Hydrolase II from Lactobacillus fermenti 36 ATCC 9338

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of N^α-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co²⁺, is essential for its activity.     

Enantioselectivity-promoting factor in enzyme-mediated asymmetric hydrolysis of enol esters

Enantioselectivity-promoting factor enhances enantioselectivity of protonation in lipase AP-catalyzed asymmetric hydrolysis of enol esters. The factor was partially purified by chromatography using Phenyl-TOYOPEARL 650M and Sephacryl S-200HR. The hydrolysis of 2-benzyl-l-cyclohexenyl acetate by PLE in the presence of the purified factor produced (R)-2-benzylcyclo-hexanone in 92% ee, while the reaction without the factor gave the racemate.     

Purification of a multiphosphorylated peptide from canine cardiac myosin heavy chains labeled in tissue culture

Partial acid hydrolysis of canine cardiac myosin heavy chains labeled with [³²P]orthophosphate in myocardial cell culture yielded a peptide having a molecular weight between 700 and 1500 and containing phosphothreonine and phosphoserine. The phosphate-rich peptide of myosin heavy chains produced by partial acid hydrolysis was purified first by Sephadex gel filtration, followed by elution with a gradient of formic acid from a Dowex ion-exchange chromatograph. Further identification of the multiphosphorylated peptide was made using high voltage electrophoresis and amino acid analyses. The data described here demonstrate that partial acid hydrolysis (time dependent) can be used to produce partially acid-stable peptides in a good yield.     

Purification and characterization of a lipase from Pichia burtonii

An extracellular lipase from Pichia burtonii was purified to homogeneity by a combination of DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. The purified enzyme preparation showed a single protein band corresponding to a molecular mass of 51 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 47 kDa on Superdex 200 gel filtration, suggesting that the enzyme was a monomeric protein. The pI was about 5.8. The optimum pH and temperature for the hydrolysis of olive oil were about 6.5 and 45°C respectively. Rapid loss of the enzyme activity was observed above 30°C in the absence of olive oil, but the addition of olive oil or trimethylolpropane diallyl ether greatly stabilized the enzyme. At 30°C, the enzyme hydrolysed Spans and Tweens as well as simple triglycerides of short- and middle-chain fatty acids. Although the enzyme cleaved all the ester bonds of triolein, it showed some preference for the outer ester bonds.     

Purification and Properties of Co2+ Requiring Heme Protein Having Lipoxygenase Activity from Fusarium oxysporum

Co²⁺-requiring heme protein having lipoxygenase activity, obtained from Fusarium oxysporum (FUSARIUM lipoxygenase) was extensively purified by ammonium sulfate precipitation, ion exchange chromatography on SP-Sephadex and gel filtration with Sephadex G–100. The final preparation achieved homogeneity by ultracentrifugation and SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated at 12,000 to 13,000 on the basis of ultracentrifugation, SDS-polyacrylamide gel electrophoresis and gel filtration. FUSARIUM lipoxygenase contained 1 mole protoheme IX per mole enzyme, required Co²⁺ as a stabilizing factor and lost activity by treatment with heat or proteases. FUSARIUM lipoxygenasecatalyzed oxidation was proved to be differrent from the well-known soybean lipoxygenasecatalyzed oxidation and hemeprotein or cobalt-catalyzed oxidations in various respects including reaction velocity, substrate specificity pI and activation energy.     

Isolation and partial characterization of glabrin, a neurotoxin from Cnestis glabra (Connaraceae) root barks

A neurotoxic compound, inhibiting protein synthesis in cell culture, was isolated in a yield of about 0.4 per cent from Cnestis glabra root barks (Connaraceae) by a five-step fractionation procedure (filtration on activated charcoal, treatment by neutral lead acetate and fractionations on Dowex 50 X 8 in H+ and NH+4 forms). The purified toxin appeared homogeneous on thin-layer and in gas chromatography. The compound has a low molecular weight (less than 500). It is heat-stable, insoluble in usual organic solvents and gives a positive reaction with ninhydrin. Acidic hydrolysis does not change its behaviour on an amino acid analyzer. Its possible amino acid nature is discussed. It is temporarily named glabrin.     

Purification and Some Properties of a Neutral α-Glucosidase from Pig Serum

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s_20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 10⁵ by thin-layer gel filtration and SDS-disc electrophoresis.

The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.