Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase : An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin1

A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg⁻¹ was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

Purification and characterization of caffeine synthase from tea leaves.

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.     

Characterization of a thermostable alpha-amylase from a thermophilic Streptomyces megasporus strain SD12

An extracellular alpha-amylase (1,4-alpha D-glucan glucan hydrolase; EC 3.2.1.1) was isolated from the cell free broth of Streptomyces megasporus SD12 grown in glucose, soluble starch and raw starch. The enzyme was purified 55-fold with a specific activity of 847.33 U mg-1 of protein and with a yield of 36% activity. The apparent molecular mass of the enzyme was 97 kDa, as estimated by SDS-PAGE. The pI of the enzyme was 5.4 and it was stable at a pH range of 5.5 to 8.5 with an optimum pH 6. The enzyme was stable upto 85 degrees C with a half life of 60 min. With soluble starch as substrate the enzyme exhibited a K(m) and kcat value of 4.4 mg ml-1 and 2335 U min-1 mg-1 of protein respectively. The major end products of starch hydrolysis were maltotriose and maltose depending on the incubation period. The production of the enzyme with agricultural wastes as substrates was 643 to 804 U min-1 mg-1 of protein in submerged fermentation whereas solid state fermentation could produce only 206 U min-1 mg-1 of protein.     

Feruloyl esterase from aspergillus sp.: purification, properties, and action on natural substrates

An enzyme active toward the methyl ester of ferulic acid was isolated from the fungus Aspergillus sp. and purified to homogeneity using ion-exchange and hydrophobic chromatography. The molecular weight of the enzyme is 42 kD, and its pI is 3.7. The enzyme has a pH optimum in the range 4-6 and a temperature optimum in the range 40 to 60 degrees C. Using a number of synthetic and natural substrates, the enzyme was identified as a feruloyl esterase. The feruloyl esterase did not hydrolyze wheat straw. Ferulic acid was detected as a product of hydrolysis of wheat bran and sugar-beet pulp. Other products were also detected after sugar-beet pulp hydrolysis.     

Purification and characterization of an alpha-L-rhamnosidase from Aspergillus nidulans

An enzyme exhibiting alpha-L-rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase was shown to be N-glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4.5-6 and 60 degrees C, had an isoelectric point of 5. With rho-nitrophenyl-alpha-L-rhamnopyranoside as the substrate it showed Km and Vmax values of 0.27 mmol l-1 and 64.6 U mg-1, respectively. The enzyme was competitively inhibited by L-rhamnose (Ki 0.3 mmol l-1). Ca2+ (2 mmol l-1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l-1) inhibited it by 63%. Substrate specificity studies showed the alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides.     

Catalytic properties of the cloned amylase from Bacillus licheniformis.

A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli. The genomic DNA of B. licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322. The transformed E. coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B. licheniformis DNA. The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch. It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C. The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4. It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage. The enzyme also hydrolyzed pullulan to panose units exclusively. In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage. The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose. The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.     

Purification and characterization of acetyl esterase from Candida guilliermondii

An extracellular acetyl esterase (EC 3.1.1.6) from Candida guilliermondii NRRL Y-17257 was purified to homogeneity by acetone precipitation and QAE sepharose anion-exchange chromatography. The enzyme was a monomer with an apparent molecular weight of 67 kDa and a pI of 7.6. It had maximum activity at pH 7.5 and at 50-60 degrees C. It was relatively stable over a pH range of 5.8-8.0 and exhibited thermal stability up to 60 degrees C. The Km and Vmax values on alpha-naphthylacetate were 2.63 mM and 213.3 micromol alpha- naphthol min-1 mg-1 protein, respectively.     

Kinetics of L-gamma-Glu-pNA hydrolysis by destabilase, the enzyme from the medicinal leech Hirudo medicinalis

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).     

A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Purification and properties of two enzymes of meta-cleaving the aromatic ring controlled by the biphenyl biodegradation plasmid pBS 241 from Pseudomonas putida

It was shown that two metapyrocatechases (EC 1.13.11.2) function in Pseudomonas putida BS893. Biphenyl degradative plasmid pBS241 carries the genes of these enzymes. The basic properties of the both enzymes, i. e., MPC1 and MPC2, were investigated. It was found that MPC1 is an enzyme with a molecular mass of 135 kD and has a heterotetrameric subunit structure (alpha 2 beta 2), being made up of two non-identical polypeptides with Mr of 34 and 22.5 kD; pI is 5.15, the pH optimum is at 8.0, a temperature optimum is at 54 degrees C. MPC2 has a molecular mass of 154 kD and possesses a homotetrameric subunit structure (alpha 4); it consists of identical polypeptides with Mr of 41 kD and has a pI of 4.95, a pH optimum at 7.5 and a temperature optimum at 60 degrees C. The substrate specificity of the enzymes was studied, and the Km and Vmax values for substituted catechols were determined. MPC1 shows a high affinity for 2.3-dihydroxybiphenyl and hydrolyzes 3-methylcatechol and catechol (but not 4-methylcatechol) at a low rate. MPC2 has a moderate affinity for catechol, 3- and 4-methylcatechols, but is incapable of cleaving 2.3-dihydroxybiphenyl. Both enzymes share in common some typical properties of metapyrocatechases. The different role of MPC1 and MPC2 in biphenyl catabolism is discussed.     

Purification and characterization of F420H2-dehydrogenase from Methanolobus tindarius.

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane-bound F420H2-dehydrogenase which was purified 31-fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2-dehydrogenase, which was yellowish, contained 16 +/- 2 mol iron and 16 +/- 3 mol acid-labile sulfur/mol enzyme. No flavin could be detected. The oxygen-stable enzyme catalyzed the oxidation of F420H2 (apparent Km = 5.4 microM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 mumol.min-1.mg-1 (kcat = 25.5 s-1). The isoelectric point was at pH 5.0. The temperature optimum was at 37 degrees C and the pH optimum at 6.8.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

Purification and characterization of extracellular beta-galactosidase secreted by supension cultured rice (Oryza sativa L.) cells

A beta-galactosidase was purified 1300-fold by lactosyl-Sepharose 4B and Sephacryl S-200 column chromatographies from the cultured medium of a rice-cell suspension. The purified enzyme appeared as 47 kD and 40 kD polypeptides on SDS-PAGE and had a specific activity of 65.1 units/mg. Optimum activity was observed at pH 3.5 and 60 degrees C. The enzyme released galactose from galactoxyloglucan and pectic galactans.     

假单胞杆菌D-海因酶的纯化及酶学性质

D-海因酶是工业上生产D-型氨基酸的关键酶,用热变性,硫酸铵沉淀及Sepharose Q fast flow,Phenyl-Sepharose fast flow,Superose 12等柱层析步骤从pseudomonas2262菌体中分离纯化了该酶,纯化倍数约为60,活力回收约为16%.该酶为同源二聚体,分子量约为109kD,亚基分子量约为53.7kD,反应最适pH为8.0,最适温度为70℃,在pH6.0～10.0和温度60℃以下稳定,该酶对巯基试剂敏感,大多数二价金属离子如镁、锰离子等能促使酶活提高,但高浓度锌离子能抑制酶活,以二氢尿嘧啶为底物的米氏常数Km=2.5×10-2mol/L.该酶的N末端10个氨基酸残基依次为MDKLIKNGTI.     

Identification of protease from Euphorbia amygdaloides latex and its use in cheese production

In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH4)2SO4 fractionation and CM-cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60 degrees C. The V(max) and K(M) values at optimum pH and 25 degrees C were calculated by means of Linewearver-Burk graphs as 0.27 mg/L min(-1) and 16 mM, respectively. The purification degree was controlled by using SDS-PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits.In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese.     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.