Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.     

A New Peptide-N 4-(acetyl-β-glucosaminyl)asparagine Amidase from Soybean (Glycine max) Seeds: Purification and Substrate Specificity

We report here the isolation and characterization of a peptide-N ⁴-(acetyl-β-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the β-aspartylglycosylamine linkage (GlcNAcβ1→Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

Occurrence and synthesis of lectin in coleoptiles of germinating wheat,rye and rice seedlings

Occurrence, synthesis and localization of lectins in coleoptiles of 3-day old seedlings of wheat, rye, barley and rice were studied by a combination of high resolution ion-exchange chromatography, in vivo labelling with ³⁵S-cysteine and immunocytochemistry. Whereas no lectin can be isolated or localized in barley coleoptile, 1.9 and 40 ng of lectin per coleoptile was obtained from wheat and rye respectively. Wheat germ agglutinin was localized in the outer layer of the wheat coleoptile and both inner and outer layers of rye coleoptile displayed a specific reaction. In rice, 250 ng of lectin is present in the coleoptile and is distributed throughout this organ. Wheat coleoptiles synthesize no lectin and rye coleoptiles synthesize minute amounts while those from developing rice seedlings incorporate reasonable amounts of ³⁵S-cysteine into lectin.Abbreviations FPLC Fast Protein Liquid Chromatography - GlcNAc N-acetylglucosamine - PBS phosphate buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Purification and characterization of novel transglutaminase from Bacillus subtilis spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37 degrees C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60 degrees C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked epsilon-(gamma-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as alphas-casein and BSA.     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

人γ－精浆蛋白的亲和纯化及其糖基化分析

目的:从人精液中提纯γ-精浆蛋白并对其生物学特性进行鉴定。 方法:应用饱和硫酸铵法从小鼠腹水中纯化出抗γ-精浆蛋白的单克隆抗体E4B7,将其共价交联到CNBr-Sepharose4B上,制备成免疫亲和层析柱,用该层析柱亲和纯化经饱和硫酸铵粗提的精液标本。纯化产物分别用SDS-PAGE、Western-blot及ELISA进行生物学特性鉴定,最后经PNGase F、Endo-H酶消化后进行糖基化分析。结果:SDS-PAGE电泳表明纯化蛋白的相对分子量约为44KD,Western-blot及ELISA鉴定显示该蛋白可与抗γ-精浆蛋白的单抗E4B7特异性结合。对纯化蛋白无论是单用Endo-H酶消化,还是同时用Endo-H酶和PNGase F酶共同消化,消化产物电泳后相对分子量均降低到34KD左右,而单独用PNGase F酶消化后相对分子量没有改变。Western-blot及ELISA鉴定显示糖苷酶处理后的纯化蛋白仍可与单抗E4B7特异性结合。结论:成功纯化出N-糖基化形式的γ-精浆蛋白,这为下一步筛选人源化抗体奠定了纯的抗原基础。     

Purification and Characterization of Novel Transglutaminase from Bacillus subtilis Spores

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37°C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60°C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked ε-(γ-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as α_s-casein and BSA.     

Purification and characterization of beta-N-acetylhexosaminidase from rice seeds

N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

The Purification and Properties of a Copper Nitrite Reductase from Haloferax denitrificans

A dissimilatory nitrite reductase from Haloferax denitrificans was purified to apparent electrophoretic homogeneity. The overall purification was 125-fold with about a 1% recovery of activity. The enzyme, which had a molecular mass of 127 kDa, was composed of a 64-kDa subunit as determined by SDS-PAGE. Although maximum activity occurred in the presence of 4 M NaCl, no activity was lost when the enzyme was incubated in the absence of NaCl. The absorption spectrum had maxima at 462, 594, and 682 nm, which disappeared upon reduction with dithionite. Diethyldithiocarbamate (DDC) was inhibitory, and the addition of copper sulfate to DDC-inhibited enzyme partially restored activity. These results suggest this enzyme is a copper-containing nitrite reductase and is the first such nitrite reductase to be described in an Archeon.     

DNA topoisomerase I from rice: Enzyme synthesis in germination, and partial purification from cultured cells

DNA topoisomerase I activity was observed in two-day-old seeds of rice when the seeds started germination at 30°. Partially purified enzyme from cultured rice cells showed maximum activity at pH 7.0 with 75 mM NaCl in the absence of ATP, and showed resistance to camptothecin and DNA-intercalating reagents. The M_r was ca 80 000 using gel permeation on a Sephacryl S-200 column. After fractionation of the homogenate from cultured rice cells by centrifugation, the activity was observed mainly in the crude nuclear fraction.     

Single-step Purification by Lectin Affinity and Deglycosylation Analysis of Recombinant Human and Porcine Deoxyribonucleases I Expressed in COS-7 Cells

Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

Effect of polyamines on ribonuclease activity of rice (Oryza sativa L.)

The RNA content and RNase activity were determined during maturation and germination of rice seeds. The RNA content reached a maximum after the 16th day from anthesis but RNase activity steadily increased up to the last stage of maturation. During germination RNA content was greatest after 24 hr and associated with a very low level of RNase activity. Maximum RNase activity was observed at 72 hr from germination and it afterwards gradually declined. During germination, exogenous application of polyamines decreased the level of RNase activity. RNase was partially purified (448-fold) from 72 hr germinated embryonic axis. Effects of polyamines and other divalent cations were observed on the purified enzyme.     

Purification and characterization of hydantoin racemase from Microbacterium liquefaciens AJ 3912

A hydantoin racemase that catalyzed the racemization of 5-benzyl-hydantoin was detected in a cell-free extract of Microbacterium liquefaciens AJ 3912, a bacterial strain known to produce L-amino acids from their corresponding DL-5-substituted-hydantoins. This hydantoin racemase was purified 658-fold to electrophoretic homogeneity by serial chromatography. The N-terminal amino acid sequence of the enzyme showed homology with known hydantoin racemases from other microorganisms. The apparent molecular mass of the purified enzyme was 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 117 kDa on gel-filtration in the purification conditions, indicating a homotetrameric structure. The purified enzyme exhibited optimal activity at pH 8.2 and 55 degrees C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

Purification and Some Properties of Active β-Amylase in Rice

An active β-amylase was purified from germinated rice seeds by precipitation with ammonium sulfate, acid treatment, chromatographies on DEAE-cellulose and DEAE-Sephadex A-50, and gel filiations on Sephadex G-75. The purified enzyme was homogeneous in disc electrophoretic analysis.

The molecular weight was estimated to be approximately 53,000 by thin-layer gel filtration and polyacrylamide gel electrophoresis. The isoelectric point was found to be pH 5.0 by disc electrofocusing.

The optimum pH was found to be in the pH range of 5.5 to 6.5. The Km value for soluble starch was 3 mg/ml. The enzyme was inhibited by sulfhydryl reagents or heavy metal ions.

The active β-amylase was oxidatively dimerized by treatment with 0.3 m ferricyanide in 3 m urea. The dimerized enzyme was thought to be one of inert β-amylases in ungerminated rice seeds.     

从海洋中分离的弧菌QY102褐藻胶裂解酶的纯化和性质研究

从马尾藻(Sargassum)表面分离到一株产生高效胞外褐藻胶裂解酶的海洋弧菌（Vibrio sp.） QY102。以褐藻胶为唯一碳源发酵培养后,发酵液上清通过0.22μm滤膜过滤、DEAESepharose离子交换和Superdex75凝胶过滤得到电泳纯的褐藻胶裂解酶。酶的性质研究表明：其分子量约为28.5kD（SDSPAGE）,反应最适温度为40℃,最适pH为7.1,Ca2+、Mg2+对酶活有促进作用,而Ni2+、Al3+、Zn2+、Ba2+对酶活有抑制作用。该酶的活性明显高于已报道的褐藻胶裂解酶,pH稳定范围广（5～10）,并且对聚甘露糖醛酸的活性高于对聚古罗糖醛酸的活性。     

Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.