Isolation,purification, and biochemical characterization of a novel water soluble protein from Inca peanut (Plukenetia volubilis L.)

A water soluble storage albumin from Inca peanut (IPA) accounted for approximately 25% (w/w) of defatted seed flour weight, representing 31% of the total seed protein. IPA is a 3S storage protein composed of two glycosylated polypeptides, with estimated molecular weights (MW) of 32800 and 34800 Da, respectively. IPA has an estimated sugar content of 4.8% +/- 0.92% (n = 6). IPA is a basic protein (pI of approximately 9.4) and contains all of the essential amino acids in adequate amounts when compared to the FAO/WHO recommended pattern for a human adult. The tryptophan content of IPA is unusually high (44 mg/g of protein), whereas the phenylalanine content is low (9 mg/g of protein). IPA is a highly digestible protein in vitro.     

Isolation and characterization of a bioactive mannose-binding protein from the Chinese chive Allium tuberosum

A mannose-binding protein was isolated from two different cultivars of the Chinese chive Allium tuberosum by extraction with 0.2 M NaCl, ammonium sulfate precipitation, and affinity chromatography on mannose agarose and fetuin agarose. It exhibited hemagglutinating activity toward rabbit erythrocytes. The lectin (agglutinin) was adsorbed on the mannose-agarose column, but not on the fetuin-agarose column. This A. tuberosum lectin (ATL) is unglycosylated, and not sialic acid binding. Lectins isolated from the two cultivars exhibited the same molecular mass of 25 kDa on gel filtration (Superose 12) and 12.5 kDa on SDS-polyacrylamide gel electrophoresis, indicating that they might be a dimeric protein composed of two identical subunits. The N-terminal amino acid sequence analysis of the lectin of various cultivars of A. tuberosum revealed that they were identical and showed 50%, or more, homology to the lectins from Galanthus nivalis (family Amaryllidaceae), Narcissus tazetta (family Amaryllidaceae), and Aloe arborescenes (family Liliaceae).     

Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.     

Isolation and characterization of an oxygen radical absorbance activity peptide from defatted peanut meal hydrolysate and its antioxidant properties

Defatted peanut meal hydrolysate (DPMH) was purified using ultrafiltration, gel filtration chromatography, and high-performance liquid chromatography. A tripeptide with strong oxygen radical absorbance capacity (ORAC) was isolated and identified as Tyr-Gly-Ser by ESI-MS/MS. It was then synthesized to measure its antioxidant properties in different systems. The ORAC value of Tyr-Gly-Ser was 3-fold higher than that of glutathione (GSH), and it displayed a stronger protective effect on linoleic acid peroxidation and H(2)O(2)-induced oxidative injury in rat pheochromocytoma line PC12 cells than GSH (p < 0.05). However, Tyr-Gly-Ser showed negligible DPPH radical scavenging activity, reducing power, and no metal chelating ability. The results suggested that Tyr-Gly-Ser displayed antioxidant activity via the hydrogen atom transfer mechanism, and the Tyr at the N-terminal was the hydrogen donor. The ORAC assay was recommended as a reliable and effective method to measure the antioxidant activity in the course of antioxidant peptide isolation.     

Isolation and characterization of a novel tannase from a metagenomic library

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.     

Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.     

Purification, identification, and characterization of peanut isocitrate lyase

Isocitrate lyase (ICL, EC 4.1.3.1) is commonly present in oil-rich seeds in catalyzing the cleavage of isocitrate to glyoxylate and succinate and plays an essential role in lipid metabolism and gluconeogenesis. When peanut kernels (Tainan 14) were germinated at 30 degrees C, the cotyledon ICL activities increased substantially in the initial 4 days, and the 4-day-germinated cotyledons were subjected to ICL purification by Tris-HCl buffer extraction, heat treatment at 55 degrees C for 1 h, (NH4)2SO4 fractionation at 25-35% saturation, DEAE-cellulose chromatography, and Sephacryl S-300 gel filtration. A single 64 kDa SDS-PAGE protein band was obtained with 7.7% recovery and 37.5-fold purity. It was identified as ICL by LC-MS/MS analyses and Mascot Search with 494 as the highest Probability Based Mowse Score (PBMS). On the basis of the sequence of the homologous ICL of Glycine max, 26% of the peptide sequences of the peanut ICL were identified. During gel filtration, separation of peanut catalase (identified by LC-MS/MS and Mascot Search with 405 as the highest PBMS) from peanut ICL was achieved. The highest measured peanut ICL enzymatic activities were obtained at 45 degrees C and pH 7.0-7.8, respectively. The enzyme activities were stable (>80%) as stored for 8 h at 30 degrees C, 15 days at 4 degrees C, or 60 days at -25 degrees C. As affected by the supplements in the reactants for activity determinations, ICL activity was not affected by glucose up to 4%, sucrose up to 5%, or ethanol up to 8.33%.     

Isolation and characterization of glyoxal-arginine modifications

5-(4,5-Dihydroxy-2-imino-1-imidazolidinyl)norvaline (1) was identified as the only product of the early reaction of arginine with glyoxal, which was slowly degraded to N(5)-[[(carboxymethyl)amino](imino)methyl]ornithine (3, N(7)-carboxymethylarginine). No other structures could be detected within a range of pH 4-8 and 20-50 degrees C in reaction conditions. The rates of formation for both products increased with pH and temperature. In equilibrium, the vincinal diol groups of 1 were 86% trans configured. The formation of 1 was reversible, as could be shown by cis-trans isomerization of the separated isomers and by regeneration of arginine in the presence of the alpha-dicarbonyl trapping reagents, o-phenylenediamine and aminoguanidine. Both 1 and 3 were converted to 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline (2) only under strong acidic conditions.     

花生品质对其蛋白质凝胶性的影响

为了探讨不同花生品种的感官品质、理化与营养品质、加工品质与其蛋白质凝胶性之间的关系,提出适宜肉制品加工凝胶型蛋白质专用花生品种的主要特性和评价方法,构建适宜加工凝胶型蛋白质花生品质评价模型。以62个花生品种制得的分离蛋白粉为原料,采用物性仪测定其分离蛋白凝胶性,分析各品种花生的品质特性与凝胶性之间的关系,采用有监督主成分分析对41个品种的品质数据建立凝胶型蛋白质花生品质评价模型,并用另外21个品种品质数据进行验证。结果显示,不同品种花生蛋白质在相同条件下制得的凝胶特性差异显著。果形、粗蛋白、粗纤维、半胱氨酸、甘氨酸、精氨酸、亮氨酸、伴花生球蛋白Ⅰ、花生球蛋白/伴花生球蛋白、23.5kDa等10个指标与凝胶性在0.05水平上关系显著,采用有监督主成分分析建立凝胶型蛋白质花生品质评价模型,验证模型的相关系数为0.937。研究结果表明,花生的品质特性显著影响花生蛋白质的凝胶性。花生粗蛋白含量、胱氨酸含量、精氨酸及伴花生球蛋白Ⅰ含量高的品种具有更好的凝胶性。通过凝胶型蛋白质花生品质评价模型可以预测该品种花生蛋白质的凝胶性,为不同花生品种的加工利用和专用品种的选择提供依据。     

Isolation and characterization of patatin isoforms.

Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented. Patatin could be separated in four isoform pools, denoted A, B, C, and D, representing 62%, 26%, 5%, and 7% of the total amount of patatin, respectively. These isoforms differed in surface charge, resulting in a different behavior on anion exchange chromatography, isoelectric focusing, native polyacrylamide gel, and capillary electrophoresis. All isoforms of the patatin family contained proteins with two molecular masses of approximately 40.3 and 41.6 kDa, respectively. The size of this difference in the molar mass (1300 Da) is on the order of one carbohydrate moiety. Despite the biochemical differences given above, no variations in the structural properties nor in the thermal conformational stability could be observed using far-ultraviolet circular dichroism, infrared, and fluorescence spectroscopy.     

Isolation and characterization of a low molecular weight peptide contained in sourdough

To investigate a sourdough-specific peptide, low molecular weight peptides were extracted from sourdough. The peptide fraction was subjected to two kinds of chromatography to separate the peptides. Reverse-phase chromatography of the peptide fraction in the sourdough showed certain specific peptides. The specific peptide fraction was further separated by gel filtration chromatography. Liquid chromatography tandem mass spectrometry analysis identified one of the peptides as VPFGVG (six-mer). This sequence was estimated to occur at the 287-292 position of a low molecular weight glutenin subunit. The peptide (designed as SDP1) was produced by proteases derived from wheat flour. SDP1 showed angiotensin-converting enzyme (ACE) inhibitory activity, and the 50% inhibitory peptide concentration (IC50) was 336 microM. It is possible that the SDP1 peptide partially confers ACE inhibitory activity in sourdough.     

一株芽孢杆菌属的苄嘧磺隆降解菌的分离鉴定和降解研究

The objectives of this study were to isolate a bensulfuron-methyl (BSM)-degrading strain of Bacillus spp. and to evaluate its effectiveness in remediation of a BSM-contaminated soil. A BSM-degrading bacterium, strain L1, was successfully isolated in this study. Strain L1 was identified as Bacillus megaterium based on its morphological, physiological, and biochemical properties, G+C content, phylogenetic similarity of 16S rDNA, and fatty acid compositions. Two experiments were used to examine BSM degradation by strain L1. When BSM was used as a sole carbon source in a mineral salt medium, the average degradation rate of BSM by strain L1 was 12.8%, which suggested that the strain was able to utilize BSM as a sole carbon and energy source. In addition, supplement of yeast extract (200 mg L-1) significantly (P ≤ 0.01) accelerated the degradation of BSM by strain L1. Almost complete degradation (97.7%) of BSM could be achieved in 84 h with addition of yeast extract. In addition, when a sterile soil was supplemented with BSM (50 mg L-l), BSM degradation rate was 94.3% in 42 d, indicating the potential of using microbes for the remediation of BSM-contaminated soils in fields.     

Isolation and partial characterization of banana starches.

Two varieties of banana green fruit growing in Guerrero, Mexico, were used for starch isolation. Chemical analysis and physicochemical and functional properties were studied in these starches. The "macho" variety presented higher starch yield than "criollo". In general, chemical compositions in both starches were similar, except in ash content, where the "criollo" variety showed a lower value than "macho". The results of freeze-thaw stability suggested that banana starches cannot be used in frozen products. Both starches presented similar water retention capacity values that increased when temperature increased. Solubility profiles showed that at low temperature "criollo" had lower solubility than "macho", but at higher temperature an inverse behavior was evident; also the solubility increased when temperature increased. Behavior similar to that for solubility was obtained in the swelling test. The banana starch studies indicate the "macho" and "criollo" varieties have different starch structures as evidenced by viscosity.     

灭多威降解菌mdw-1的分离和降解特性研究

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30-C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.     

不同制备方法对花生蛋白功能性质的影响(简报)

该文研究了不同制备方法对花生浓缩蛋白功能性的影响,以期为不同制备方法制得的花生浓缩蛋白在食品中的广泛应用提供理论支持。以脱脂花生蛋白粉（DPF）为原料,通过等电沉淀、乙醇浸提、等电沉淀与乙醇浸提相结合及碱溶酸沉技术制备花生浓缩蛋白,并分别测定其蛋白功能性（蛋白溶解性、吸水性、持油性、乳化能力及乳化稳定性、起泡能力及泡沫稳定性、凝胶性质）。结果表明：碱溶酸沉技术制备的蛋白溶解性、起泡能力及泡沫稳定性最好;而乙醇浸提制备的蛋白吸水性、持油性和凝胶性质要显著性的高于其他方法制备的蛋白产品的;不同方法制备的花生浓缩蛋白的乳化稳定性均明显低于对照（DPF）,尤以碱溶酸沉技术制备的最低。因此可知,乙醇浸提制备的蛋白适用于对吸水性、持油性和凝胶性质要求较高的食品中;碱溶酸沉技术制备的蛋白适用于对起泡能力要求较高的食品中。     

Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans

Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.     

Alcalase酶水解花生蛋白制备花生短肽的研究

对Alcalase水解花生蛋白制备花生短肽过程中,酶用量、底物浓度、pH值、反应温度、反应时间等影响因素进行了系统地研究,建立了短肽得率及水解度与各种影响因素的回归模型;在此基础上,结合实际生产确定出了Alcalase酶解花生蛋白的最适条件为pH值8.0,水解温度54℃,底物浓度4%,酶用量3480 U/g,水解时间106 min。在此条件作用下,体系中短肽得率为79.08%,水解度为17.08%,短肽平均链长为5.85,平均分子量为661.5。     

Isolation and characterization of a novel Arabidopsis thatiana mutant that requires a high concentration of boron

We isolated a novel Arabidopsis thaliana mutant line that requires high levels of boron (B) for normal growth. Line 8–21 was identified from ethyl methanesulfonate-mutagenized M2 population. When grown in medium containing 3 μm or less B in the form of boric acid, the fresh weights of aerial portions of the mutant were about a half of those of the wild type, but in that containing 300 μm B, the growth appeared normal. The mutant plants did not shown any difference in root growth from the wild-type plants in the range of B concentrations tested. When grown with 30 μm B, the B concentration in shoots of the line 8–21 was similar to that in the wild-type, suggesting that the mutant could not utilize B efficiently. Line 8–21 was not allelic to bor1-1 (Noguchi et al., Plant Physiol., 115, 901–906, 1997). A significant portion of F2 plants from the crosses between the wild-type and the mutant grew poorly on a low B media, suggesting segregation of the mutation.     

Isolation and characterization of a mycorrhiza helper bacterium from rhizosphere soils of poplar stands

If mycorrhizal formation could be enhanced by co-inoculation with mycorrhiza helper bacteria (MHB) which promote rapid root colonization by specific ectomycorrhizal fungi, this would be of advantage to the poplar forest industry. A number of poplar rhizobacterial strains were isolated from 11 regions of the eastern China. Four of the isolates, SY15, DZ18, HLJ4, and PY10, were characterized as MHB potential strains based on their positive effect on growth of ectomycorrhizal fungi Pisolithus tinctorius (Pers.) and Lactarius insulsus (Fr.). Under greenhouse conditions, one of the bacterial isolate, DZ18, significantly promoted the poplar trees growth and ectomycorrhizal colonization of P. tinctorius and L. insulsus on Populus deltoides Marsh. In contrast, the other three isolates SY15, HLJ4, and PY10 promoted fungal growth in vitro experiments but did not enhance ectomycorrhizal (ECM) formation in the greenhouse experiment. Therefore, it was concluded that DZ18 can be considered as an MHB strain. DZ18 was identified as Bacillus sp. based on morphological, physiological, and biochemical analyses in combination with analysis of 16S rDNA gene sequences.