Galactoglucomannan Oligosaccharides (GGMO) from a molasses byproduct of pine ( Pinus taeda ) fiberboard production

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a β-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.     

红松种子壳多酚物质的提取及抗氧化特性

该文选取超声波辅助提取法,采用单因素试验及响应面设计,研究了超声波提取时间、温度、乙醇浓度及料液比对红松种子壳中多酚类化合物提取的影响。并利用Fenton体系、1,1-二苯基-2-苦苯肼(DPPH·)体系及邻苯三酚体系测定其提取成分的抗氧化活性。结果表明,松壳中多酚成分提取的最佳工艺条件为:超声提取时间2.80h、料液比1∶26.36(g/mL)、乙醇浓度42.86%、提取温度80℃,多酚得率7.96mg/g。在此优化条件下,红松种子壳提取物对羟自由基(甲基紫法)、DPPH·自由基及超氧阴离子均具有较强清除作用,半数抑制浓度分别为6.67×10-4、2.34×10-2、3.97mg/mL。     

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.     

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.     

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 trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

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 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 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.     

Purification and partial characterization of chickpea 2S albumin

A chickpea 2S albumin has been purified by solubilization in 60% methanol and ion-exchange chromatography. Under denaturing conditions it is composed of two peptides of 10 and 12 kDa. Native molecular mass determined by gel filtration chromatography is 20 kDa. Amino acid composition shows that it is rich in sulfur amino acids, mainly cysteine with 4.6% of the total. On the other hand, it has antinutritional characteristics of being allergenic for chickpea-sensitive individuals and inhibitory against porcine chymotrypsin with a lesser degree toward trypsin. The results of interest from a nutritional point of view are discussed.     

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 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.     

Chemical composition of essential oils from needles and twigs of balkan pine (Pinus peuce grisebach) grown in Northern Greece

The composition of essential oils from twigs and needles of Balkan pine (Pinus peuce Gris.) grown in northern Greece was investigated. The compounds were identified by using GC-MS analysis. The twig oil was rich in alpha-pinene (7.38%), beta-pinene (12.46%), beta-phellandrene (26.93%), beta-caryophyllene (4.48%), and citronellol (12.48%), and the needle oil was rich in alpha-pinene (23.07%), camphene (5.52%), beta-pinene (22.00%), beta-phellandrene (6.78%), bornyl acetate (9.76%), beta-caryophyllene (3.05%), and citronellol (13.42%). The mean oil yield was 2.85% for twigs and 0. 57% for needles.     

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.     

Short-term effects of thinning on soil respiration in a pine (Pinus tabulaeformis) plantation

Respiration was measured at daytime during the growing seasons (May–October) of 2011 and 2012 in a young Pinus tabulaeformis plantation with heavy, medium and light intensity thinning and unthinned control plots in Shanxi province in northern China. Soil temperature, moisture, fine root biomass, amounts of soil organic C and litterfall biomass were also measured. We found that immediately following thinning treatments, soil respiration increased by 8 %–21 % compared with the unthinned control plots during both growing seasons. Thinning significantly affected soil respiration and soil temperature with different thinning intensities, while there were no significant differences in soil moisture among the various treatments. During the growing seasons, the soil respiration rates were positively correlated with the soil moisture: the 19.4 %–54.0 % variation in soil respiration rates in the four thinning regimes are explained by the changes in soil moisture. Meanwhile, a positive correlation was found between soil temperature and soil respiration rates at all sites. The best fitting model with temperature and moisture explained 44.3 % of the variation in soil respiration in the high thinning treatment, 27.6 % in the light thinning treatment, 18.6 % in medium thinning and in the control sites during the measuring periods. Overall, soil respiration is better predicted by soil moisture, soil organic C, live fine root biomass and soil temperature when data are pooled for all thinning treatments over the two growing seasons. The best regression model explained 74.7 % of the total variation in soil respiration over the different thinning intensities for the two sampling periods.     

11S and 7S globulins of coconut (Cocos nucifera L.): purification and characterization

Total globulins extracted with 0.4 M NaCl in buffer from coconut endosperm separated into two peaks on gel filtration: peak I corresponding to 11S globulin or cocosin and peak II to 7S globulin with native molecular weights of 326 000 and 156 000, respectively. The percent composition of total globulins was estimated to be 11S, 86% and 7S, 14%. On SDS-PAGE, cocosin resolved into two closely migrating bands at approximately 34 000 (acidic polypeptide) and another set of 2 bands at 24 000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. The 7S globulin consisted of three bands of 16 000, 22 000, and 24 000. Three isoforms of cocosin were identified after anion exchange chromatography. Cocosin, but not the 7S, was found to have disulfide bonds. Using periodic acid-Schiff's reagent, all of the bands of cocosin on SDS-PAGE were positive for carbohydrate. However, when con A-peroxidase was used, only the basic polypeptide stained positively for carbohydrate. For the 7S globulin, no carbohydrate group was detected using the PAS and con A-peroxidase tests. The 7S globulin was easily extracted with 0.10-0.15 M NaCl, whereas cocosin was extracted with 0.35 M NaCl. The N-terminal amino acid sequences of the 34 k band and 24 k band of cocosin were SVRSVNEFRXE and GLEETQ, respectively, and that of the 7S was EQEDPELQK.     

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 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.     

原始红松林穿透雨和树干茎流养分特征研究

对小兴安岭原始红松林穿透雨和树干茎流中的K,Ca,Mg,Mn,Fe共5种养分元素进行了测定,结合水量分配比例,计算了各元素在森林中的净输入量。结果表明:⑴原始红松林穿透雨和树干茎流中,除Fe元素外,所有浓度值均有树干茎流>穿透雨>大气降雨;从各元素平均浓度来看,Ca元素最高,Fe元素最低,其排序为:Ca>K>Mg>Mn>Fe。⑵大气降雨年平均浓度中,K元素变动系数最大,为1.514,Ca元素最小,为0.827;原始红松林穿透雨、树干茎流年平均浓度中变动系数最大的元素分别为Mn,Mg(或Fe);变动系数最小的元素分别为Ca,Mn;各元素年平均浓度的变异系数均为大气降雨>穿透雨>树干茎流。⑶原始红松林各元素净输入量排序为:K>Ca>Mg>Mn>Fe,且Fe元素的净输入量为负值。