Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Isolation and characterization of a phenolic antioxidant from the Pacific oyster (Crassostrea gigas)

Using an oxygen radical absorbance capacity (ORAC) assay, antioxidant activity was detected in the ethanol extract of the Pacific oyster, which was purified by sequential extraction with organic solvents. The ethyl acetate fraction showed the strongest antioxidant activity and was further purified, yielding a single compound [as assessed by thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (HPLC)]. This compound was identified as 3,5-dihydroxy-4-methoxybenzyl alcohol on the basis of (1)H and (13)C nuclear magnetic resonance (NMR), heteronuclear multiple-bond correlation (HMBC), and electrospray ionization-mass spectrometry (ESI-MS) spectral analyses, a conclusion that was confirmed by chemical synthesis. The concentration of the compound was 6.7 mg/100 g of whole oyster meat wet weight. This amphiphilic antioxidant retarded the copper-mediated oxidation of low-density lipoproteins (LDLs) and the generation of thiobarbituric acid reactive substances. Furthermore, the compound showed substantial antioxidant activity using the ORAC and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays compared to natural antioxidants. Although the same compound was previously found in brown algae, its presence in other organisms and antioxidant activity are reported here for the first time.     

秘鲁鱿鱼皮明胶-壳聚糖复合膜的性能与结构表征

为考察壳聚糖对鱿鱼皮明胶膜的改性效果,将壳聚糖添加到明胶溶液中,考察壳聚糖的添加量对鱿鱼皮明胶复合膜机械性能、水蒸气透过率、透光性及其结构的影响。结果表明,壳聚糖能有效改善鱿鱼皮明胶膜的有关性能指标,当鱿鱼皮明胶溶液与壳聚糖溶液以60∶40(v/v)比例混合,制得复合膜的断裂伸长率相比单一明胶膜下降,但其抗拉伸强度、透光率和水蒸气阻隔能力分别比单一明胶膜提高了652%、11%和48%;差示扫描量热仪、红外及扫描电镜分析结果显示,壳聚糖能与鱿鱼皮明胶相互作用,形成结构致密的均相体系,提高复合膜的热变性温度。综上可知,鱿鱼皮明胶与壳聚糖之间具有良好的相容性,壳聚糖是一种较理想的明胶膜改性材料。本试验结果为鱿鱼皮明胶作为食品保鲜膜的应用提了供依据。     

响应面法优化秘鲁鱿鱼(Dosidicus gigas)肌肉盐溶蛋白的提取和凝胶形成条件

探讨了秘鲁鱿鱼肉盐溶蛋白的提取及凝胶形成条件对其凝胶特性的影响。采用响应面分析法,研究KCl浓度、pH值和低温加热时间对鱿鱼肉盐溶蛋白凝胶保水性和质构特性的影响。结果显示:(1)提取液KCl浓度对保水性影响极其显著,对硬度影响显著;提取液pH值对保水性影响显著;低温加热时间对硬度和粘性影响显著;(2)在分析各因素显著性及其交互作用的基础上,确定鱿鱼肉盐溶蛋白提取和凝胶形成的适宜条件为:KCl浓度0.16 mol·L-1、pH 7.10、40℃加热55min,此时凝胶保水性、硬度、粘性分别达到88.00%、510.00g、-11.00g,与模型预测值相符。     

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis)

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.     

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.     

秘鲁鱿鱼吸盘环齿脱离工艺条件优化及其结构特性分析

为了实现秘鲁鱿鱼加工副产物的高值化利用,以鱿鱼加工废弃物中的鱿鱼吸盘为原料,以脱齿率为指标,采用不同蛋白酶对其进行脱环齿试验,以确定最适蛋白酶,在单因素试验基础上,利用响应面法对其酶解工艺进行优化,对鱿鱼吸盘环齿主要成分进行测定,并通过红外光谱(IR)、扫描电子显微镜(SEM)和差示扫描量热法(DSC)对鱿鱼吸盘环齿(SRT)的结构特性进行分析。结果表明,秘鲁鱿鱼吸盘环齿最适酶解脱离工艺条件为酶添加量1 700 U·g^-1、酶解温度43℃、酶解时间34 min,此条件下样品脱齿率为96.2%,蛋白质含量为86.75%。红外光谱分析证明,鱿鱼吸盘环齿主要成分为蛋白质,不含有甲壳素;扫描电镜观察发现秘鲁鱿鱼吸盘环齿微观构造为平行管状模块组成的多孔结构;热稳定分析显示其在118.6℃处有熔融峰,具有一定的热塑性。同时,鱿鱼吸盘环齿还具有一定的耐酸耐碱性。本研究结果为进一步开发利用秘鲁鱿鱼吸盘环齿提供了理论依据。     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

添加剂对秘鲁鱿鱼肌原纤维蛋白热诱导凝胶特性的影响

为探讨添加剂及其添加量对秘鲁鱿鱼肌原纤维蛋白热诱导凝胶特性的影响,以秘鲁鱿鱼为原料,在单因素试验基础上选择4种添加剂(壳聚糖、转谷氨酰胺酶、卡拉胶、海藻酸钠),通过响应面法建立肌原纤维蛋白凝胶硬度和添加剂用量之间的响应模型。结果表明,4种添加剂的最适添加量分别为:壳聚糖0.40%、转谷氨酰胺酶0.50%、卡拉胶0.90%、海藻酸钠0.25%,在此条件下秘鲁鱿鱼肌原纤维蛋白热诱导凝胶硬度达最大值859.85 g,保水性为96.34%。此复合添加剂能显著改善秘鲁鱿鱼肌原纤维蛋白的凝胶特性。研究结果为生产高品质的秘鲁鱿鱼凝胶制品提供了理论依据。     

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.     

Purification and characterization of the 7S vicilin from Korean pine (Pinus koraiensis)

Pine nuts are economically important as a source of human food. They are also of medical importance because numerous pine nut allergy cases have been recently reported. However, little is known about the proteins in pine nuts. The purpose of this study was to purify and characterize pine nut storage proteins. Reported here is the first detailed purification protocol of the 7S vicilin-type globulin from Korean pine (Pinus koraiensis) by gel filtration, anion exchange, and hydrophobic interaction chromatography. Reducing SDS-PAGE analysis indicated that purified vicilin consists of four major bands, reminiscent of post-translational protease cleavage of storage proteins during protein body packing in other species. The N-terminal ends of vicilin peptides were sequenced by Edman degradation. Circular dichroism (CD) and differential scanning calorimetry (DSC) analyses revealed that pine nut vicilin is stable up to 80 degrees C and its folding-unfolding equilibrium monitored by intrinsic fluorescence can be interpreted in terms of a two-state model.     

Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps

A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.     

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous)

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Purification and characterization of a cysteine protease inhibitor from chum salmon (Oncorhynchus keta) plasma

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.     

Purification and characterization of molybdenum species from Medicago sativa (alfalfa) grown on mine tailings

Extraction, purification, and identification procedures were developed for the chemical investigation of molybdenum (Mo) in freeze-dried aerial portions of alfalfa (Medicago sativa L.) collected from a reclaimed tailings pond at the Highland Valley Copper mine in British Columbia. The purification procedures were guided by ICP and colorimetric analyses. The methods included development of an efficient aqueous extraction protocol, sample cleanup by partitioning against n-butanol, and filtration through diatomaceous earth. Further purification was achieved by anion-exchange chromatography, elution with aqueous NaCl, and desalting by gel filtration. Final purification of the Mo-containing fraction was carried out using preparative anion-exchange HPLC. Molybdenum was found to be present in its purified form as the molybdate anion (MoO(4)(2-)) primarily on the basis of multinuclear ((1)H, (13)C, and (95)Mo) NMR studies.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Purification and characterization of gelatinase-like proteinases from the dark muscle of common carp (Cyprinus carpio)

Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.     

Purification and characterization of windmill palm tree (Trachycarpus fortunei) peroxidase

High peroxidase activity was demonstrated to be present in the leaf of several species of cold-resistant palms. Histochemical studies of the leaf of windmill palm tree (Trachycarpus fortunei) showed the peroxidase activity to be localized in hypoderma, epidermis, cell walls, and conducting bundles. However, chlorophyll-containing mesophyll cells had no peroxidase at all. The leaf windmill palm tree peroxidase (WPTP) was purified to homogeneity and had a specific activity of 6230 units/mg, RZ = 3.0, a molecular mass of 50 kDa, and an isoelectric point of pI 3.5. The electronic spectrum of WPTP with a Soret band at 403 nm was typical of plant peroxidases. The N-terminal amino acid sequence of WPTP was determined. The substrate specificity of WPTP was distinct from that of other palm peroxidases, and the best substrate for WPTP was 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid). The palm peroxidase showed an unusually high stability at elevated temperatures and high concentrations of guanidine.     

Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase

Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.