Purification and characterization of an extracellular proline iminopeptidase from Arthrobacter nicotianae 9458

An extracellular proline iminopeptidase, with a molecular mass of about 53 kDa, was purified from Arthrobacter nicotianae 9458 and characterized. The enzyme had temperature and pH optima of 37 degrees C and 8.0, respectively, was completely inactivated by heating for 1 min at 80 degrees C and showed highest activity on Pro-pNA. The proline iminopeptidase was characterized by activity at low temperature, NaCl concentrations up to 7.5% and by high sensitivity to pH values 6.0, serine enzyme inhibitor PMSF and divalent cations, Fe2+, Sn2+, Cu2+, Zn2+, Hg2+, Co2+ and Ni2+. The extracellular proline iminopeptidase from A. nicotianae 9458 was able to hydrolyze proline-containing peptides at the pH, temperature and NaCl concentration typical of the surface of smear-ripened cheese and may contribute to proteolysis of these cheeses during ripening.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Purification and characterization of aminopropionaldehyde dehydrogenase from Arthrobacter sp. TMP-1

Aminopropionaldehyde dehydrogenase was purified to apparent homogeneity from 1,3-diaminopropane-grown cells of Arthrobacter sp. TMP-1. The native molecular mass and the subunit molecular mass of the enzyme were approximately 20,5000 and 52,000, respectively, suggesting that the enzyme is a tetramer of identical subunits. The apparent Michaelis constant (K(m)) for 1,3-diaminopropane was approximately 3 microM. The enzyme equally used both NAD(+) and NADP(+) as coenzymes. The apparent K(m) values for NAD(+) and NADP(+) were 255 microM and 108 microM, respectively. The maximum reaction rates (V(max)) for NAD(+) and NADP(+) were 102 and 83.3 micromol min(-1) mg(-1), respectively. Some tested aliphatic aldehydes and aromatic aldehydes were inert as substrates. The optimum pH was 8.0-8.5. The enzyme was sensitive to sulfhydryl group-modifying reagents.     

Purification and characterization of a novel sialidase from a strain of Arthrobacter nicotianae

The nonpathogenic strain Arthrobacter nicotianae produces two sialidase isoenzymes, NA1 and NA2, with molecular masses of 65 kDa and 54 kDa, respectively, as determined by 10% SDS-polyacrylamide gel electrophoresis. NA1 and NA2 exhibit maximum activities at pH 4 and 5, and both show clear thermal optima at 40 degrees C. They are stable at temperatures up to 50 degrees C. The critical temperatures (T (c) = 50 degrees C and 51 degrees C) for the two isoenzymes were determined by fluorescence spectroscopy and correlate well with the temperatures of melting (T (m) = 49 degrees C and 48 degrees C), determined by CD spectroscopy. The isoenzymes are less stable against denaturation with Gdn.HCl, and the free energy of stabilization in water was calculated to be 7.6 and 8.0 kJ mol(-1), respectively. The specific activity (K (m) value) toward glucomacropeptide as a substrate was calculated to be 0.126 mM for NA1 and 0.083 mM for NA2.     

Purification and properties of an endo-inulinase from an Arthrobacter sp.

Extracellular endo-inulinase of Arthrobacter sp. S37 was purified 63-fold, giving a single band on PAGE with activity staining. The Mr was estimated as 75 kDa by SDS-PAGE. The first 31 amino acids of the N-terminal sequence was determined. The endo-inulinase hydrolyzed inulin mainly into inulo-triose (F3), inulo-tetraose (F4) and inulo-pentaose (F5) optimally at pH 7.5 and 50°C. © Rapid Science Ltd. 1998     

Purification and characterization of major extracellular proteinases from Trichophyton rubrum.

Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).     

Divergent pathways for δ-aminolevulinic acid synthesis in two species of Arthrobacter

Abstract Secretion of coproporphyrin III by suspensions of Arthrobacter photogonimos and A. globiformis facilitated analysis of the paths of synthesis of δ-aminolevulinic acid, the precursor of tetrapyrroles. Sensitivity of coproporphyrin accumulation to gabaculine and incorporation of ¹⁴C from [1-¹⁴C]glutamate indicated that suspensions of A. photogonimos synthesized δ-aminolevulinic acid from glutamate by the widespread C5 pathway. In contrast, A. globiformis cells switched from predominantly the C5 pathway for δ-aminolevulinic acid synthesis in early exponential phase cultures to δ-aminblevulinic acid synthase in stationary phase cultures.     

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase

AIMS: The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. METHODS AND RESULTS: Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. CONCLUSIONS: Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.     

Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.     

十二节杆菌胞外脂肪酶的纯化和性质研究

十二节杆菌发酵得到的胞外脂肪酶,在5L发酵罐经过34h培养,最高酶活可达75 U/mL。通过硫酸铵梯度沉淀和疏水层析纯化,脂肪酶纯化26倍,总得率24.3%。SDS-PAGE显示脂肪酶分子量为33 kD,脂肪酶在40℃和pH 7.0时酶活力最高,同时在24℃经过48h仍保持一半左右的活力。该脂肪酶的酶活受K ,Mg2 激活,而受Zn2 与Co2 的抑制。     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.