Amine Oxidases of Microorganisms

The amine oxidase was found to be formed in mycelia of fungi when they were grown on monoamines or diamines as sole nitrogen sources. The maximal formation of enzyme was observed in the initial stage of growth, then the enzyme disappeared semilogarithmically. Other sources of nitrogen, such as ammonia, nitrate, urea and amino acids, were fully inactive for the enzyme formation. Furthermore, ammonia repressed the enzyme formation by fungi. The amine oxidase of fungi resembled in substrate specificity the monoamine oxidase of animal tissues. The enzyme oxidized preferentially aliphatic monoamines of C₃–C₆. Agmatine and histamine were also oxidized but in lower rates. Benzylamine was well oxidized by the enzymes of Aspergillus niger and Penicillium chrysogenum, but not by the enzymes of Monascus anka and Fusarium bulbigenum. Polyamines were not oxidized by the fungal enzymes.     

Rat Stem-Cell Leukemia Gene Expression Increased during Testis Maturation

Histamine oxidase (EC class 1.4.3) was found in cells of Arthrobacter globiformis IFO 12137 (ATCC 8010) grown on histamine. The enzyme purified to a specific activity of 9.4units/mg had a purity of at least 80%. The enzyme oxidized histamine with concomitant formation of H₂O₂. Phenylethylamine, dopamine, aromatic monoamines, and aliphatic mono- and diamines were oxidized at lower rates. Cu²⁺-chelators inhibited the enzyme, indicating that the enzyme is Cu²⁺-dependent. Carbonyl-blocking reagents also inhibited the enzyme. The enzyme catalyzed the Nitro Blue Tetrazolium/glycinate reaction, which is characteristically catalyzed by quinones and quinoproteins. These results strongly suggest that the enzyme contains a quinonoid cofactor.     

Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on β-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (α₂β₂) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the α-subunit (42.3-kDa subunit) and the β-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the β-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and β-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, β-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 μM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).     

Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. no. 203

Polyethylene glycol (PEG) 4000-utilizing bacterium no. 203 was identified as a Flavobacterium species. 2, 6-Dichlorophenol-indophenol (DCIP)-dependent PEG dehydrogenase was constitutively formed in nutrient broth, glucose and PEG media. However, the enzyme formation was repressed in the presence of an excess amount (over 0.25%) of PEG 400 or 1000. PEG dehydrogenase was purified approximately 34 fold by precipitation with ammonium sulfate, solubilization with benzalkonium chloride, chromatography with DEAE-Toyopearl 650 M and hydroxylapatite and gel filtration on Toyopearl HW-55. The molecular weight of the purified PEG dehydrogenase was calculated to be approximately 2.20 × 10⁵, a value which seemed to consist of four subunits with the same molecular weight of 5.70 × 10⁴. The enzyme was stable below 40°C and in the pH range of 7.0 and 8.0. The optimum pH and temperature of the activity were around 8.0 and 40°C, respectively. The enzyme reduced DCIP and coenzyme Q₁ and Q₂. PEG dehydrogenase showed activity toward various PEG molecules (dimer-PEG 20,000). The apparent K_m values for PEG 400, 1000, 4000 and 6000 were about 1.0, 1.7, 2.8 and 5.9 mM, respectively. The enzyme oxidized primary aliphatic alcohols of C₃–C₁₂, the corresponding aldehydes of C₃–C₇, aromatic alcohols and aldehydes, diols, etc. The enzyme was inactive on ethylene glycol, glycerol, secondary alcohols and sugar alcohols. The enzyme activity was strongly inhibited by sulfhydryl agents or heavy metals and 1, 4-benzoquinone. The purified enzyme showed absorption apectrum similar to that of PEG 6000 dehydrogenase which has already been reported to be a quinoprotein. The prosthetic group of the enzyme was extracted with methanol and identified as PQQ from its prosthetic group capability for glucose dehydrogenase and the fluorescence spectrum.     

Gas-liquid Chromatographic Determination of Carbohydrates in Tobacco

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight carbonyl compounds examined. The enzyme reaction on oxidized PVA resulted in a rapid decrease in viscosity, a fall of pH, and production of carboxylic acids. The enzyme, therefore, is considered to be an oxidized PVA hydrolase.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to an oxidized PVA hydrolase previously purified from another fraction adsorbed on SP-Sephadex at pH 7.0 from the PVA-degrading enzyme activities [Agric. Biol. Chem., 45, 63 (1981)]. The relations between these two oxidized PVA hydrolases are discussed.     

Synthesis and Biological Activity of (±)2′,3′-Dihydroabscisic Acid

An enzyme which catalyzes the oxidation of poly(vinyl alcohol) (PVA) has been purified from a fraction adsorbed to DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a culture broth when the culture was grown in a minimal medium where PVA served as a sole source of carbon and energy. The enzyme was separated from a coexisting oxidized PVA hydrolase by dye-ligand chromatography on Matrex Gel Blue A. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoreses in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 40,000 and has an isoelectric point of 4.5. The amino acid composition of the enzyme has been determined and found to have no histidine. The N- and C-terminal amino acid residues are both alanine. The enzyme solution is pink and shows absorption maxima at 276, 364, and 469 nm. One atom of non-heme iron has been detected per molecule in the enzyme.

The enzyme catalyzes the oxidation of PVA and also of various low molecular weight secondary alcohols to the corresponding ketones with the production of H₂0₂ and the consumption of 0₂. The molar ratio of these ketones, H₂0₂ and 0₂ is 1:1:1. The most effective electron acceptor is 0₂, while 2,6-dichlorophenolindophenol and nitro blue tetrazolium also serve as the acceptor with efficiencies to 0₂ of about 31 and 16%, respectively. The enzyme is, therefore, considered to be a secondary alcohol oxidase.

The enzyme is most active at pH 7.0 and at 45°C and is stable between pH 5.0 and 9.0 and at temperatures below 45°C. The activity is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to a secondary alcohol oxidase previously isolated from another fraction adsorbed on SP-Sephadex at pH 7.0 of the PVA-degrading enzyme activities [Agric. Biol. Chem., 43, 1225 (1979)]. The relations between these two secondary alcohol oxidases are discussed.     

Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22

The lipase was partially purified by ion exchange chromatography and gel filtration column chromatography, and was characterized from Geobacillus stearothermophilus AH22 strain. The lipase was purified 18.3-folds with 19.7% recovery. The lipase activity was determined by using p-nitrophenyl esters (C₂–C₁₂) as substrates. The K_m values of the enzyme for these substrates were found as 0.16, 0.02, 0.19 and 0.55?mM, respectively, while V_max values were 0.52, 1.03, 0.72 and 0.15?U?mg^?1. The enzyme showed maximum activity at 50?°C and between pH 8.0 and 9.0. The enzyme was found to be quite stable at pH range of 4.0–10.0, and thermal stability between 50 and 60?°C. It was found that the best inhibitory effect of the enzyme activity was of Hg²⁺. The inhibitory effect as orlistat, catechin, propyl paraben, p-coumaric acid, 3,4-dihydroxy hydro-cinnamic acid was examined. These results suggest that G. stearothermophilus AH22 lipase presents very suitable properties for industrial applications.     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

Occurrence and Some Properties of Cellulase in the Filtrates of Conidiospores and Mycelia of Pyricularia oryzae Cavara

Occurrence of cellulase activity was demonstrated in the filtrates of germinating conidiospores and growing mycelia of P. oryzae. Activity and some properties of cellulase in the filtrate of mycelia grown on rice plant powder as carbon source were compared among various strains.

Cellulase activity (C₁ and C_x enzymes; cellulose and carboxymethylcellulose as substrates, respectively) in the filtrate of germinating conidiospores was detected in the pathogenic T–l (Ken 53–33) strain as well as nonpathogenic 0 (THU 3 × 1) strain of P. oryzae. The activity was higher in the former than the latter strains. Cellulase activity (C_x enzyme) in the filtrate of growing mycelia was detected in the four strains used, T–l (Ken 53–33), C–3 (N 87), N–1 (H373), and 0 (THU 3 × 1). Cellulase activity (C_x enzyme) in the filtrate of mycelia was optimal at pH 5.0 and 40°C, and stable up to 40°C. Their properties did not differ significantly except for the pH-activity curve at alkaline side among various strains; but cellulase activity (C₁ enzyme) was found to be correlated with their pathogenicity except for the case of C–3 strain.     

Purification and Some Properties of An Aldehyde Oxidase from Streptomyces Rimosus ATCC10970

Summary An aldehyde oxidase was purified from a cell-free extract of Streptomyces rimosus ATCC10970 to an electrophoretically homogeneous state. The molecular mass of the native enzyme was estimated to be 150 kDa by a gel filtration. SDS-polyacryamide gel electrophoresis showed that the enzyme consisted of three non-identical subunits with molecular masses of 79, 39 and 23 kDa. The absorption spectrum revealed a distinctive feature as an enzyme belonging to the xanthine oxidase family with maxima at 277, 325, 365, 415, 450, 480, and 550 nm. A variety of aliphatic and aromatic aldehydes were oxidized, but nitrogen-containing heterocyclic compounds were not. Among the substrates tested, n-heptanal was most rapidly acted on. Its optimum pH and temperature were pH 7.0 and 30 °C, respectively.     

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier^?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.     

Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

Crystallization and Characterization of Polyamine Oxidase from Penicillium chrysogenum

Polyamine oxidase was purified and crystallized with an overall yield of 35% from mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 column chromatographies. The crystalline enzyme was homogeneous, as judged by disc gel electrophoresis and ultracentrifugation. The sedimentation coefficient (s_{20, w}⁰) of the enzyme was determined to be 6.9S, and diffusion coefficient (D_{20, w}) to be 4.2 × 10^?7 cm² sec^?1. The enzyme showed a molecular weight of about 160,000 by gel filtration method and ultracentrifugal analysis, and it was composed of two identical subunits. The enzyme was a flavoprotein with absorption maxima at 275, 375 and 450 nm. The prosthetic group was identified to be FAD. The enzyme oxidized spermine, and slightly oxidized spermidine. Diamines and monoamines were not oxidized.     

Studies on Pectic Enzymes of Microorganisms

Endo-polygalacturonase (endo-PG) of Aspergillus saitoi was purified through ammonium sulfate fractionation, Amberlite IRC-50 column chromatography, and several combinations of Sephadex column chromatography.

The purified endo-PG, which was almost homogeneous ultracentrifugally and electrophoretically, had the sedimentation constant of 2.2 S and the absorption maximum at 277 mμ. Its optimum pH and temperature were 4.8~5.0 and 45°C, respectively, and it was most stable between pH 4.0 and 6.0, but over 90% of the activity was lost at 50°G for 10 min.

The purified enzyme was a typical endo-PG, and hydrolyzed about 60% and 17% of glycosidic linkage of polygalacturonic acid and pectin, respectively. This enzyme preparation had no pectinesterase, trans-eliminase, and apple juice-clarifying activities, but macerated potato tuber slices singly.     

Substrate specificity of copper-containing plant amine oxidases

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Biochemical and Stability Properties of Recombinant Human MnSOD

The light absorption spectral properties of recornbinant human MnSOD. which contains an N-terminal additional methionyl residue, were investigated as a function of pH in the range 4.5–10.5. Whereas the extinction coefficient, ?_M at the UV maximum (282 nm) was essentially independent of pH, the ?_M values of the visible spectrum maximum (482 nm) displayed a bell-shaped dependence with a plateau between pH 6.5 and B. Those spectral changes were reversible and the enzymatic activity was not affected by exposure to buffered solutions at 25°C in the pH range 5–10.5. The stability of MnSOD was determined between 25 and 60°C at two different pH: 6.5 and 8.2. The enzyme was found to be considerably more stable at pH 6.5 than at pH 8.2, both toward aggregation and degradation. The gel permeation properties of MnSOD were investigated: the enzyme is a tetramer, with a subunit of 22.2 kD; however. it elutes from a Superose 12 column (Pharmacia) with an apparent molecular weight of ～60kD. Under dissociative conditions (such as guanidine-HCI). molecular weights corresponding to the dimer and monomer could also be demonstrated. It thus appears that the tetramer adopts a non-globular shape. which causes the deviation from the Stokes radius corresponding to its molecular weight.     

Bacterial Monoamine Oxidases

A procedure for obtaining crystalline preparations of tyramine oxidase of Sarcina lutea has been developed. The procedure included fractionation with ammonium sulfate, treatment with protamine sulfate and separation by column chromatographies on DEAE-cellulose, hydroxylapatite and sephadex G-150. The specific activity of enzyme was increased 5,700~ 6,000-fold through the procedure, over the crude cell extract. Crystals were prepared from solutions of the purified enzyme by adding solid ammonium sulfate. The crystals appeared as minute, highly refractive needles, with a bright yellow color.

With the use of crystalline preparations of tyramine oxidase of Sarcina lutea, substrate and inhibitor specificities of the enzyme were investigated. The enzyme oxidized tyramine and dopamine at almost the same rates. Other monoamines, diamines, polyamines and amino acids were not oxidized at all. The oxidation of tyramine proceeded as follows: Tyramine+O₂+H₂O→p-Hydroxyphenylacetaldehyde +NH₃+H₂O₂. Ammonia and hydrogen peroxide were formed in stoichiometric amounts.

The enzyme was not inhibited by carbonyl reagents, such as hydroxylamine, hydrazine, semicarbazide and isoniazid, but was inhibited by p-CMB and iproniazid.     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Cloning and characterization of a novel thermostable amidase,Xam, from Xinfangfangia sp. DLY26

Objective

Identification and characterization of a novel thermostable amidase (Xam) with wide pH tolerance and broad-spectrum substrate specificity.

Results

Xam was identified from non-thermophilic Xinfangfangia sp. DLY26 and its acyl transfer activity was investigated. Recombinant Xam was optimally active at 60 °C and pH 9.0. The enzyme had a half life of 18 h at 55 °C and maintained more than 60?% of its maximum activity in the range of pH 3.0–11.0. Additionally, Xam exhibited broad substrate specificity towards aliphatic, aromatic, and heterocyclic amides.

Conclusions

These unique properties make Xam a promising biocatalyst for production of important hydroxamic acids at elevated temperatures.