Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp. LP7315

A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on SDS-PAGE with a molecular mass of 59 kDa. Its hydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze di- and triglycerides. MGL was found to be stable even after 1-h incubation at 65 degrees C. The optimum pH for monopalmitin hydrolysis was approximately 8. The hydrolytic activity depended not only on temperature and pH but also on the type of monoglyceride used. MGL also catalyzed monoglyceride synthesis at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in the glycerol phase with a low water content. The synthetic reaction proceeded at a constant rate for approximately 24 h and approximately reached an equilibrium after 48 h of reaction. The initial rate and equilibrium yield of the synthetic reaction depended on the type of fatty acid used as the substrate.     

Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis

Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.     

Purification and thermostability of beta-galactosidase (lactase) from an autolytic strain of Streptococcus salivarius subsp. thermophilus

beta-Galactosidase from an autolytic strain of Streptococcus salivarius subsp. thermophilus was purified 109-fold to near homogeneity. The yield of purified enzyme was 41% and the specific activity was 592 o-nitrophenyl beta-D-galactopyranoside U/mg at 37 degrees C. Two isozymes were present, but only one subunit was detected, having a mol. wt of 116,000. Enzyme stability was 37-83 times greater in milk than in buffer in the range 60-65 degrees C. At 60 degrees C the half-life in milk was 146 min. Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was approximately 0.5. The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer. In milk the activation energy for lactose hydrolysis was 35.1 kJ/mol.     

A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

A thermostable histamine oxidase (EC 1.4.3.-) was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The enzyme was purified about 715-fold over the cell free extracts with a yield of 55% by ammonium sulfate fractionation and various column chromatographies. The purified enzyme was homogeneous on polyacrylamide gel-electrophoresis (native-PAGE). When the enzyme was kept at 65 degrees C and 70 degrees C for 10 min, the activity was fully stable at 65 degrees C and decreased to 9% of the initial level at 70 degrees C. The enzyme was very thermostable. The optimum pH for histamine oxidase activity was found to be at 9.0, and the enzyme was stable over the pH range of 6 to 9. The purified enzyme showed a single protein band on SDS-PAGE and its molecular mass was estimated to be about 81 kDa. The enzyme showed potent activity toward histamine, whereas it was inactive toward putrescine, cadaverine, spermine, and spermidine. Histamine oxidase was inhibited by N,N-diethyldithiocarbamate (DDTC). The inactive enzyme was restored with Cu2+ to 65% of the initial activity, but Cu+ did not enhance the enzyme activity. It is suggested that Cu2+ is essential for expression of histamine oxidase activity. The enzyme was a copper-containing protein having one atom of copper per mol of the enzyme protein as a result of atomic absorption analysis. The N-terminal amino acid sequence of the purified enzyme was different from that of histamine oxidase from Arthrobacter globiformis IFO12137.     

Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans

3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Laccase and Mn-peroxidase production by Coriolus hirsutus strain 075 in a jar fermentor

The white-rot fungus Coriolus hirsutus strain 075 excretes considerable amounts of laccase and Mn-peroxidase into culture broth over a brief production time. The effects of agitation speed, temperature, aeration and inoculum amount on laccase production using a 10-l fermentor were studied. The optimum fermentation conditions were a 15% inoculum, an aeration rate of 0.88 vvm, an agitation speed of 160 rpm, and a temperature of 28 degrees C. By optimizing the fermentation conditions, the laccase activity reached 80+/-3 U/ml in 3 d and the purified enzyme output was 30 mg/l. The laccase and Mn-peroxidase were purified by means of isoelectrofocusing and ion-exchange chromatography. The pIs of the laccase isoenzymes were 4.2 and 4.5. Mn-peroxidase had only one isoenzyme with a pI of 3.2. The optimum pH was 4.5 for laccase with syringaldazine as the substrate and 5.0-5.3 for Mn-peroxidase with Mn(+2) and H2O2 as the substrates. The laccase and Mn-peroxidase retained 50% of their activities at 50 degrees C after 55 h and 12 h of incubation time, respectively.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Thermal inactivation of Escherichia coli O157:H7 in cow manure compost

Rates of inactivation of a five-strain mixture of green fluorescent protein-labeled Escherichia coli O157:H7 in autoclaved and unautoclaved commercial cow manure compost with a moisture content of ca. 38% were determined at temperatures of 50, 55, 60, 65, and 70 degrees C. Trypticase soy agar with ampicillin was determined to be the best medium for the enumeration of heat-injured and uninjured cells of green fluorescent protein-labeled E. coli O157:H7. The results obtained in this study revealed that in autoclaved compost, E. coli O157:H7 reductions of ca. 4 log CFU/g occurred within 8 h, 3 h, 15 min, 2 min, and < 1 min at 50, 55, 60, 65, and 70 degrees C, respectively. At 65 and 70 degrees C, considerably less time was required to kill the pathogen in unautoclaved compost than in autoclaved compost. Decimal reduction times (D-values) for autoclaved compost at 50, 55, 60, 65, and 70 degrees C were 137, 50.3, 4.1, 1.8, and 0.93 min, respectively, and D-values for unautoclaved compost at 50, 55, and 60 degrees C were 135, 35.4, and 3.9 min, respectively. Considerable tailing was observed for inactivation curves, especially at 60, 65, and 70 degrees C. These results are useful for identifying composting conditions that will reduce the risk of the transmission of E. coli O157:H7 to foods produced in the presence of animal fecal waste.     

Purification and characterisation of a glutaminase from Debaryomyces spp.

A glutaminase was purified from the cell-free extract of Debaryomyces spp. CECT 11815 by protamine sulphate treatment and several chromatographic procedures including anion exchange chromatography and gel filtration. The purified enzyme consisted of two subunits, with molecular masses of 65 and 50 kDa, respectively. Activity was optimal at 40 degrees C and pH 8.5, and the Km value for L-glutamine was 4.5 mM. The glutaminase exhibited activity against L-gamma-Glu-methyl ester, L-gamma-Glu-hydrazide, and L-albiziin, while L-asparagine, CBZ-L-Gln, CBZ-L-Gln-Gly, glutathione, L-gamma-Glu-pNA and L-gamma-Glu-AMC were not hydrolysed. The enzyme was not affected by PMSF, DTT and EDTA. However, the enzyme was inhibited by sulfhydryl group reagents, DON, L-albizziin, L-asparagine and high concentrations of L-glutamine and ammonium, while L-aspartate did not affect the activity. Phosphate and acetate did not produce any significant effect on the glutaminase activity, but it was slightly stimulated by lactate and borate.     

INDUCTION, PROPERTIES AND APPLICATION OF XYLANASE ACTIVITY FROM SCLEROTINIA SCLEROTIORUM S2 FUNGUS

Extracellular xylanase activity was produced in a submerged culture by Sclerotinia sclerotiorum S2 fungus, on wheat bran as an inducing substrate. The enzyme was partially purified and biochemically characterized. The optimum pH and temperature for the activity were 5.5 and 65C, respectively. The xylanase was stable over a pH range of 4.0–9.0 and retained more than 75% of its activity after incubation for 24 h without substrate. The enzyme was stable at 50C and 60C for 80 min of incubation at pH 5.0, while the half‐life was 20 min at 70C. The novel xylanase activity was useful as an aid in orange juice clarification. The clarification was observed with concomitant production of reducing sugars (0.350 mg/mL from juice) after 24 h of incubation of juice with the xylanase. Xylanase aided juice clarification, which resulted in a 27% decrease in insoluble materials.     

Partial purification and some properties of phi C2(W) lysin, a lytic enzyme produced by phage-infected cells of Streptococcus lactis C2

The lytic enzyme present in phi C2(W) lysates was isolated by means of ion-exchange chromatography and further purified by gel filtration and ultrafiltration. The phage enzyme had an apparent pH optimum of 6.5-6.9 and was rapidly inactivated at temperatures greater than 47 degrees C. The apparent temperature optimum was 37 degrees C and Q10 and Ea values over the range 22-32 degrees C were 2.5 and 69.2 kJ/mol respectively. Monovalent and divalent cations activated the enzyme. Reduced -SH groups on the enzyme were required for lytic activity. Gel filtration revealed a mol. wt of approximately 46000. Strain-dependent differences in sensitivity of group N lactic streptococci to lysin were found. Group D streptococci were also lysed. Strains of three species of Leuconostoc, two species of Lactobacillus, one strain of Escherichia coli and of Micrococcus lysodeikticus were apparently resistant. Analysis of cell wall degradation products gave results which were consistent with the lysin having the specificity of an N-acetylmuramidase.     

Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates

Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes. Heat inactivation experiments were conducted using an immersed coil apparatus. Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey. GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey. Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG. The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene. Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey. Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05. A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested.     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus

We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100000 x g. Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography. The native enzyme depicted a molecular mass of 67 kDa by gel filtration. This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively. The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane. This study indicated that K. marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

Purification and characterization of fenitrothion hydrolase from Burkholderia sp. NF100

The organophosphorus pesticide hydrolase was purified to homogeneity from Burkholderia sp. NF100 by detergent extraction of the cell membrane fraction, anion-exchange, chromatofocusing, and gel filtration chromatographies. The purified enzyme had a molecular mass of 55 kDa and a pI 5.8, and the hydrolase activity was strongly inhibited by EDTA, dithiothreitol (DTT), Hg2+ and 1,10-phenanthroline. The optimum pH and temperature for the enzyme activity were 8.0 and 40 degrees C, respectively. The enzyme hydrolyzed five organophosphorus pesticides.     

Purification and characterization of an extracellular beta-agarase from Bacillus sp. MK03

A new beta-agarase was purified from an agarolytic bacterium, Bacillus sp. MK03. The enzyme was purified 129-fold from the culture supernatant by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The purified enzyme appeared as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Estimation of the molecular mass by SDS-PAGE and gel filtration gave values of 92 kDa and 113 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed no homology to those of other known agarases. The optimum pH and temperature for this enzyme were 7.6 and 40 degrees C, respectively. The predominant hydrolysis product of agarose by this enzyme was neoagarotetraose, indicating the cleavage of beta-1,4 linkage. This enzyme could hydrolyze neoagarohexaose to produce neoagarotetraose and neoagarobiose; it could not hydrolyze these products. The enzyme digested agarose by endo-type hydrolysis.     

Properties of a thermostable beta-galactosidase from a thermophilic Bacillus: comparison of the enzyme activity of whole cells, purified enzyme and immobilised whole cells.

The properties of a β-galactosidase from a thermophilic Bacillus have been studied and compared with the properties of the enzyme contained in whole cells and in cells entrapped in a polyacrylamide gel matrix. The partially purified enzyme appears to be optimally active at a temperature of 60°C and pH 6.0. The enzyme activities from whole cells and immobilised whole cells have slightly different temperature optima (being 75 and 65°C respectively) and pH optima (being 6.2 and 6.6 respectively). The enzyme activity from whole cells is significantly more thermostable than that of the partially purified enzyme; the half-lives being 125 and 22 min respectively at 70°C. In other properties studied, the enzyme activities of the three sources are similar. The thermostability of the enzyme and the broad pH range over which it is active makes this enzyme suitable for use in the dairy industry.