Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize -sorbose to 2-keto- -gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the α-subunits of those dehydrogenases. The ability to convert -sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl₂ and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl₂ and PQQ are essential for enzymatic activity.     

Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum

(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.     

Purification and characterization of malate dehydrogenase from Corynebacterium glutamicum

The malate dehydrogenase (MDH) (EC 1.1.1.37) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 8) matched the sequence (residues 2 to 9) of the MDH from C. glutamicum (GenBank accession no. CAC83073). The molecular mass of the native enzyme was 130 kDa. The protein was a homotetramer, with a 33-kDa subunit molecular mass. The enzyme was almost equally active both for NADU and NADPH as coenzyme on the bases of the k(cat) values at pH 6.5 which is the optimum pH for the both coenzymes. Plotting of the logarithms of the 1/Km, k(cat), and k(cat)/K(m) values with respect to oxalacetate against pH lead to speculation that imidazolium is possibly a functional group in the active center of the enzyme. Citrate activated the enzyme in the oxidation of malate to oxalacetate and inhibited it in the reverse reaction.     

樟绒枝霉中一种低分子量木聚糖酶的纯化及性质研究

本文研究了樟绒枝霉（Malbranchea cinnamonmea）S168中一种低分子量木聚糖酶（McXyn25）的纯化和酶学性质。采用硫酸铵沉淀和DEAE-52阴离子柱层析两步纯化,得到电泳级纯酶,分子量为25ku。该酶的最适pH为8.0,在pH 4.5¹1.0范围内稳定;最适温度为65℃,在60℃以下具有较高的稳定性。McXyn25表现出严格的底物特异性,在纸浆漂白方面具有较好的应用前景。此外,McXyn25能够水解木聚糖产生低聚木糖,且无木糖产生,说明该酶适合应用于低聚木糖生产。      

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 the alcohol dehydrogenase with a broad substrate specificity originated from 2-phenylethanol-assimilating Brevibacterium sp. KU 1309

A novel 2-phenylethanol dehydrogenase has been purified from a soil bacterium Brevibacterium sp. KU 1309. The enzyme was purified about 1400-fold to homogeneity, and found to be a monomeric enzyme of apparent 39 kDa. The enzyme had broad substrate specificity and catalyzes a reversible oxidation of various primary alcohols to aldehydes. The enzyme required NAD+, but not NADP+ as a cofactor. Thus, the enzyme was classified into a group of NAD+-dependent primary alcohol dehydrogenase. The activity was inhibited by Cu2+, Ni2+, Ba2+, Hg2+ and p-chloromercuribenzoate. The enzyme is expected to be applicable as an effective biocatalyst in the oxidation of various alcohols.     

Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU 311

An oxidase catalyzing the conversion of glycolaldehyde to glyoxal was purified to the homogeneous state from Paenibacillus sp. AIU 311, and its properties were revealed. This enzyme was specific to glycolaldehyde and glyceraldehyde, and the reaction rates to other alcohols and aldehydes were less than 6% of that of glycolaldehyde. The Km values for glycolaldehyde and glyceraldehyde were estimated to be 13.2 and 7.5 mM, respectively. The glycolaldehyde oxidation was optimum at pH 6.5 and 50 degrees C. The molecular mass of this enzyme was 49 kDa, and it consisted of two identical subunits of 24 kDa. The NH2-terminal sequence was not homologous to those of alcohol oxidases. This is the first report of an oxidase exhibiting high specificity to a hydroxy group of aldehyde alcohols.     

Purification and characterization of NAD-specific 6-phosphogluconate dehydrogenase from Leuconostoc lactis SHO-54

The 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Leuconostoc lactis SHO-54 was purified with an overall yield of 38% and a specific activity of 140.0 units/mg protein. The enzyme had a tetrameric structure and a molecular mass of 32.8 kDa. The amino acid composition of the purified enzyme was determined, and the enzyme contained no sulfhydryl amino acids. The K(m) values for 6-phosphogluconate and NAD were 0.95 mM and 0.32 mM, respectively.     

Isolation and characterization of MELt gene from Torulaspora delbrueckii IFO 1255

Torulaspora delbrueckii IFO 1255 is a melibiose-fermenting strain in Torulaspora species. From the genome of strain IFO 1255, we obtained a 770 bp fragment by PCR with oligonucleotides synthesized based on the MEL genes of Saccharomyces cerevisiae and its related species. The region encompassing the 770 bp fragment was cloned by inverse PCR and sequenced. The nucleotide sequence revealed an open reading frame of 1422 bp encoding a 474 amino acid protein with a molecular weight of 52 360. The similarity of the presumed mature protein to Saccharomyces species and Zygosaccharomyces cidri alpha-galactosidases was 69.7-73.2% and 58.4%, respectively. The phylogenetic relationship between these species is discussed. The sequence is deposited in the DDBJ/EMBL/GenBank database under Accession No. AB027130.     

一种米曲霉蛋白酶的分离纯化及酶解特性研究

对米曲霉固态发酵所产蛋白酶分离纯化,采用硫酸铵盐析、DEAE-FF层析、Butyl-HP层析和Superdux 7510/300GL凝胶层析得到一种电泳纯的蛋白酶,SDS-PAGE显示分子量大小为27 ku左右。以酪蛋白为底物时,该蛋白酶Km=1.23 g·L-1,Vm=27.03μg·m L-1·min-1,最适反应条件为50℃,p H9.0。该蛋白酶对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低;对牛胰岛素B链上-Phe-Val-,-Cys-Gly-,-Glu-Ala-和-Arg-Gly-组成的肽键有较强的切割能力,酶切位点较多,对疏水性氨基酸具有较高的选择性,为米曲霉所产蛋白酶在食品上的应用提供有力的参考。      

Purification and characterization of a cationic peroxidase from artichoke leaves

Peroxidases are part of a large group of enzymes associated with cell wall biosynthesis, response to injury, disease, resistance and wound repair. Among peroxidase isoenzymes, a soluble cationic peroxidase (ALSP), not yet described, has been partially purified and characterized from artichoke leaves. The enzyme was shown to be a glycoprotein with a molecular weight of 51 000 and an isoelectric point of 9. The substrate specificity of the ALSP is characteristic of class III (guaiacol‐type) peroxidases. The ALSP was partially purified by ammonium sulfate precipitation, gel filtration, affinity chromatography, anionic exchange high‐performance liquid chromatography and isoelectrofocusing. The increase in specific activity was 43 times compared to the crude extract as estimated by the guaiacol assay. Three ALSP fragments were sequenced by tandem mass spectrometry de novo sequencing method. Copyright © 2007 Society of Chemical Industry     

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 a diacetyl reductase from Leuconostoc pseudomesenteroides

A diacetyl reductase (acetoin:NAD⁺ oxidoreductase EC 1.1.1.5) was purified to homogeneity from a cell-free extract of Leuconostoc pseudomesenteroides by sequentially using anion exchange chromatography, hydrophobic interaction chromatography and gel-filtration. The enzyme was optimally active for the reduction of diacetyl at pH 5.5, while optimum activity for the oxidation of meso-2,3-butanediol by the enzyme was at pH 7.5. The temperature optimum of the enzyme was 40°C. The molecular mass of the enzyme was 26.91, 30 and 95 kDa, as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and gel-filtration, respectively, indicating that the native enzyme exists as a trimer or tetramer. The enzyme used only NADH as coenzyme for the reduction of diacetyl, 2,3-pentanedione, pyruvic acid methyl ester and methyl glyoxal; NADH and NADPH functioned equally well as coenzyme for the reduction of acetoin by the enzyme. The enzyme oxidized meso-2,3-butanediol and (2S, 3S)-(+)-2,3-butanediol but not (2R, 3R)-(+)-2,3-butanediol. The apparent K_m for the reduction of diacetyl, 2,3-pentanedione and acetoin were 5.1, 5.57 and 0.34 mm, respectively.     

Purification and characterization of rice alpha-glucosidase, a key enzyme for alcohol fermentation of rice polish

Alpha-glucosidase, a key enzyme for nuka-sake brewing, was purified from Oryza sativa cv. Yamadanishiki, which is widely used for sake brewing. The molecular weight of the purified enzyme was 95 kDa. The optimum pH and temperature were 4.5 and 55 degrees C, respectively. The substrate specificity differed from that of Oryza sativa cv. Shinsetsu, which is a variety of rice consumed as a cereal. The extraction of alpha-glucosidase from the rice was stimulated by lactic acid, which suggests that lactic acid plays an important role not only in preventing bacterial contamination, but also in stimulating the parallel fermentation that occurs in nuka-sake brewing.     

麻鸭脂肪氧合酶的分离纯化及其性质研究

采用硫酸铵沉淀、离子交换层析、凝胶色谱等方法对麻鸭脂肪氧合酶(Shelduck Lipoxygenase)进行了纯化。结果显示,麻鸭LOX粗提液经60%⁸0%硫酸铵沉淀、DEAE离子交换层析、Superdex75凝胶过滤、SOURCE15Q离子交换层析后可达电泳纯,麻鸭LOX比活力为85714.3U/mg,纯化倍数98.64,经SDS PAGE分析麻鸭LOX的分子质量约为62 kDa。麻鸭LOX与大豆LOX的酶学性质差异较大,麻鸭LOX的最适温度为30℃,最适pH为6.0,且热稳定性和pH稳定性均优于大豆LOX。同时,Zn2+、Ca2+对麻鸭LOX的激活作用明显,而Mn2+对麻鸭LOX具有强烈的抑制作用。     

谷氨酸棒杆菌S9114中谷氨酸脱氢酶的初步研究

本研究以谷氨酸生产菌种S9114 为材料 ,对GDH的辅酶专一性、最适温度、最适pH和动力学参数进行了研究 ,结果发现谷氨酸生产菌S9114 细胞内含有两种GDH ,其GDH不仅能以NADPH为辅酶 ,而且能以NADH为辅酶 ,其Km值分别为 12 .4mmol/L和 1.5 8× 10 -3 mmol L ,Vmax分别为2 .4 8μmol min .ml和 5 .2 5 μmol min .ml,其最适反应温度为 4 2℃和 82℃ ,最适反应pH分别为 7.5和8.0 ,并对热比较稳定。经DEAE柱层析分析也表明谷氨酸生产菌S9114 细胞内同时存在两种GDH。     

Inducible production of alcohol oxidase and catalase in a pectin medium by Thermoascus aurantiacus IFO 31693

Thermoascus aurantiacus showed the best growth on medium containing pectin as a carbon source. The enzyme involved in the production of catalase in the fungus was alcohol oxidase. Formaldehyde dehydrogenase and formate dehydrogenase, in addition to alcohol oxidase and catalase, were detected in the cells grown on pectin. Alcohol oxidase was alkali resistant (pH 7 to 11), and was comparatively heat stable (55 degrees C).     

Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

乳酸片球菌细菌素的分离纯化及理化性质

将乳酸片球菌接种于MRS培养基,37℃静置培养17h产生细菌素。根据细菌素在pH6.0时与菌体的吸附力最强,pH2.0时吸附力最弱,通过调节pH和离心来分离纯化细菌素,得率为22.80%,比活为4.526×104AU/mg;细菌素的理化性质实验表明,细菌素经120℃处理20min仍保留70.08%的活性;在pH2～9范围内20℃处理3h抑菌活性稳定,当pH≥9时活性逐渐降低;经胃蛋白酶、胰蛋白酶、木瓜蛋白酶和蛋白酶K处理后均无抑菌活性。实验表明,该细菌素耐热、耐酸、耐碱,对蛋白酶敏感,具有开发为安全、天然的食品防腐剂的良好前景。     

Purification and characterization of a novel glutamyl aminopeptidase from chicken meat

A novel glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was purified from chicken meat by ammonium sulfate fractionation, ethanol fractionation, heat treatment, and successive column chromatographies of DEAE-Sepharose CL-6B and Sephadex G-200. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 55,000 and 550,000 by SDS-PAGE and Sephadex G-200 column chromatographies, respectively. This enzyme hydrolyzed Glu- and Asp-, but not Leu-, Arg-, and Ala-2-naphthylamide (-2NA) at all. The optimum pH and temperature for hydrolysis of Glu-2NA was 7.5. and 70°C, respectively. Reducing agents such as cysteine and dithiothreitol inhibited the activity of this enzyme at concentrations of 1 mM. However, the activation by Ca(2+) and the inhibition by amastatin were not observed.