Purification and characterization of pyrophosphatase from bighead carp (Aristichthys nobilis)

Pyrophosphatase (PPase, EC 3.6.1.1) responsible to the hydrolysis of pyrophosphate (PPi) in muscle was purified from bighead carp (Aristichthys nobilis), and characterized in detail herein for the first time. PPase was extracted with 20 mmol/L Tris-HCl buffer (pH 7.0) containing 0.05 mol/L KCl, followed by heat treatment and ammonium sulfate precipitation. Then it was purified by deithylaminoethyl-cellulose (DE52) and Sephacryl S-300 chromatography, subsequently resulted in a 114.5-fold purification. The molecular mass of the PPase was defined to be 50 kDa with two subunits. The optimum pH of the purified PPase was around 8.0, and its optimum temperature was confirmed to be 50 °C. Magnesium ion was necessary to the activity of PPase. An excess of PPi over Mg²⁺ resulted in inhibition of PPase. When the Mg²⁺/PPi molar ratio was 1:1, the maximal enzyme activity was obtained. In addition, PPase converted PPi to orthophosphate (Pi) stoichiometrically with a K_m of 1.98 mmol/L under the condition of 5 mmol/L Mg²⁺. Ethylene diamine tetraacetate (EDTA) activated the activity of PPase at low concentration, however, it consumingly did inhibit the enzyme activity at high concentration.     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp (Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.     

Purification and partial characterization of polyphenol oxidase from the flower buds of Lonicera japonica Thunb.

The purification and partial enzymology characteristics of polyphenol oxidase from Lonicera japonica (LjPPO) were studied in this paper. The crude enzyme solution was purified in turn by ammonium sulfate, dialysis, and DEAE-cellulose ion-exchange chromatography after preliminary treatments. Purification resulted in 31-fold enrichment and its molecular weight was estimated to be ∼49 kDa exhibited on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The pH for optimal conditions of LjPPO was 7.5, and the temperature was 25 °C, in addition, the inhibitive effects of inhibitors were enhanced positively with increasing of the concentration. Moreover, crude enzyme solution showed diphenolase activity toward catechol, l-dopa and chlorogenic acid rather than monophenolase and triphenolase activity, and the best substrate was catechol because of the highest V_max/K_m value. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided theoretical basis for the prevention of enzymatic browning of L. japonica during processing.     

Purification and characterization of soluble invertases from suspension-cultured bamboo (Bambusa edulis) cells

An alkaline invertase (IT I) and an acid invertase (IT II) were purified from the soluble fraction of suspension cultured bamboo cells. Both purified invertases were homogeneous as examined by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and were identified as β-fructofuranosidases able to attack the β-fructofuranoside from the fructose end. With respect to sucrose hydrolysis, the optimal pHs were 7.0 and 4.5 for IT I and IT II, respectively. The Km’s were 10.9 and 3.7 mM. The IT I and IT II molecular masses were 240 and 68 kDa, respectively, as estimated by gel filtration. The isoelectric points were 4.8 and 7.4. IT I was a homotetrameric enzyme activated by bovine serum albumin (BSA). IT II was a monomeric enzyme activated by BSA, concanavalin A (ConA) and urease. Both isoforms were significantly inhibited by heavy metal ions Ag⁺ (5 mM) and Hg²⁺ (1 mM), and mercaptide forming agent ρ-chloromercuribenzoic acid (PCMB; 0.5 mM).     

Preparation,preliminary characterization and immunostimulatory activity of polysaccharide fractions from the peduncles of Hovenia dulcis

The peduncles of Hovenia dulcis, containing abundant nutrients and having a taste like a combination of raisin, clove, cinnamon and sugar, have been consumed as fruits and used as traditional herbal medicine for a long time in China. Up to date, little information is available about the polysaccharides from peduncles of H. dulcis (HDPS) and their potential bioactivity. In this study, three purified fractions were prepared by sequential purification of crude HDPS through ion-exchange chromatography and gel-filtration chromatography. The three fractions of HDPS-1, HDPS-2 and HDPS-3 were found to be homogeneous heteropolysaccharides mainly composed of rhamnose, arabinose, galactose and galacturonic acid with an average molecular weight of 235, 70 and 53 kDa, respectively. HDPS-3 was quite different from HDPS-1 and HDPS-2, as it contained much higher content of galacturonic acid (40.5%). In vitro immunostimulatory activity evaluation revealed that all the three fractions could significantly stimulate the proliferation of splenocytes and enhance phagocytosis, nitric oxide production and acid phosphatase activity of peritoneal macrophages, which suggested that HDPS had a potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent.     

Purification,preliminary structural characterization and in vitro antioxidant activity of polysaccharides from Acanthus ilicifolius

Crude Acanthus ilicifolius (A. ilicifolius) polysaccharides (CAIP) were obtained by hot water extraction and deproteinated. Two major polysaccharide fractions, the neutral A. ilicifolius polysaccharide (NAIP) and the acid A. ilicifolius polysaccharide B (AAIP-B), were isolated from CAIP by chromatography using DEAE-Sepharose Fast Flow and Sepharose CL-6B. The molecular weights (M_w) of NAIP and AAIP-B, determined by high performance gel-filtration chromatography (HPGFC), were 11,775 and 23,161 Da, respectively. AAIP-B contained 51.23% uronic acid, characteristic of a pectin-type hetero-polysaccharide. Analysis of the neutral monosaccharide composition indicated that NAIP contained high proportions of arabinose, galactose and glucose. However, AAIP-B was mainly composed of rhamnose, arabinose and galactose. Their structure properties were confirmed by FT-IR.     

Biochemical characterization of l-arabitol 2-dehydrogenase from Pantoea ananatis

Two oxidoreductases, XDH and LAD, were found in the same operon that was involved in sugar metabolism in Pantoea ananatis. LAD, whose endogenous substrate was unknown, was recombinantly prepared and biochemically analyzed. Consequently, LAD was identified as l-arabitol 2-dehydrogenase and its substrate specificity was complementary to that of XDH.     

Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds

A plant lectin isolated in its pure state from the Egyptian seeds of Pisum sativum (PSL) produced two bands in SDS–PAGE (5.53 and 19.3 kDa; i.e. α and β chain) but one peak by gel filtration chromatography on Sephadex G-100, corresponding to 50 kDa, i.e., a dimeric structure of two monomers, each consisting of one α and one β subunit. PSL is a glycoprotein bound with glucose (2 mol/mol of protein) and stabilized by 2 atoms of each of Ca²⁺ and Mn²⁺ per molecule of protein. It highly agglutinated human, rabbit and rat erythrocytes but weakly agglutinated chicken erythrocytes, while no agglutination occurred with sheep erythrocytes. Hemagglutination was markedly affected by acidic pH, but was heat stable below 60 °C for 30 min. Among the various tested sugars, PSL agglutination was most inhibited by mannose. PSL is rich in hydroxyl amino acids while totally lacking sulfur amino acids. PSL inhibited the growth of Aspergillus flavus, Trichoderma viride and Fusarium oxysporum.     

Isolation and characterization of galactomannan from Dimorphandra gardneriana Tul. seeds as a potential guar gum substitute

A galactomannan was obtained from mature seeds of Dimorphandra gardneriana Tul., the plant from which rutin is extracted. The galactomannan extraction was based on manual separation of the endosperm, water dissolution, centrifugation and precipitation with ethanol. The galactomannan yield obtained (31%) was similar to values reported for other Brazilian seeds and to that of guar gum. The polysaccharide from D. gardneriana seeds (GalDG) was characterized by gas–liquid chromatography (GLC), gel permeation chromatography (GPC), rheology and also by ¹³C and ¹H nuclear magnetic resonance (NMR). The monosaccharide composition in weight % was mannose 64.2, galactose 34.7 and glucose 1.1. Small amounts of protein and uronic acid were found, values being 1.75 and 2.8% (w/w), respectively. The mannose/galactose ratio of GalDG (1.84) is similar to values reported for galactomannans extracted from other Brazilian seeds, and is the M/G value closest to that of guar gum (1.6–1.8). The intrinsic viscosity of galactomannan from D. gardneriana (8.7 dL/g), in water at 25 °C, is lower than the [η] value of guar gum, but the absolute viscosity of the GalDG in aqueous solution at concentrations of 0.1 and 1% (w/v) is higher. The aqueous solution at 1% (w/v) behaves as a pseudoplastic fluid, but a Newtonian behavior was noted for the solution at 0.1%. The high average molar masses, M_w of 3.9 × 10⁷ g/mol and M_n of 1.9 × 10⁷ g/mol, determined by GPC are probably due to molecular aggregation. ¹³C and ¹H NMR spectra (DEPT 135 and HSQC) of GalDG solutions in D₂O were recorded. The patterns of mannose substitution in GalDG and guar gum are similar.     

Chemical characterization of Chinese chive seed (Allium tuberosum Rottl.)

Chinese chive seeds (Allium tuberosum Rottl.) (grown in China) were investigated. Density, thousand-grain weight, and hectolitre weight of seeds were 1.27 g/cm³, 4.9 g, and 71 kg/100 l, respectively. The results showed that Chinese chive seeds contained high amounts of oil (15.8%), dietary fibre (18.2%) and crude protein (12.3%). Oil of seeds was composed of 10.1% saturated and 90.0% unsaturated fatty acids. Linoleic(69.1%) and palmitic (7.0%) were the most abundant unsaturated and saturated fatty acids, respectively. Chinese chive seeds contained 4.5 mg/kg of thiamin, 2.8 mg/kg of riboflavin and 55.1 mg/kg of niacin. The mineral contents of the seed of A. tuberosum, for iron, calcium and zinc, were 580 mg/kg, 1328 and 80.8 mg/kg, respectively. Analysis of the amino acid content of Chinese chive seed revealed that it was a rich source of the essential amino acids, isoleucine, tryptophan and lysine. The study revealed that Chinese chive seeds had high levels of nutritionally important components, such as oil, minerals and essential amino acids.     

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 °C; it was stable up to 60 °C for 1 h and in the pH range 3.0–9.5. To elucidate the enzyme’s proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin.     

Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system

Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P₁₀ and Polyhedrin promoters in the pFastBac™ dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb^I) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb^I from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb^I had insect specific glycan structures that differed from their mammalian counterparts, mAb^I similarly interacted with CD64 (FcγRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.     

Isolation and characterization of natural melanin derived from silky fowl (Gallus gallus domesticus Brisson)

Water-insoluble melanins derived from silky fowl (SF-melanin) were isolated using an enzymatic extraction procedure. The yields of the pigments from periosteum, ovary or testis, trachea, skin and muscle, were 21.3, 13.7, 10.2, 1.1, and 1.0 mg/g, respectively, on a fresh tissue basis. The isolated pigments were identified as melanins according to the Electron Paramagnetic Resonance Spectroscopy spectra. Using synthetic melanin as a calibration, their physicochemical and morphological properties were further characterized by X-ray photoelectron spectroscopy (XPS) and advanced imaging technology. Elemental composition analysis by XPS revealed that the main component of the SF-melanin was eumelanin. The morphological study showed that (1) the SF-melanin displayed ellipsoidal melanosomes which was regarded as representative of the natural material; and (2) the SF-melanin maintained its natural integrity. In conclusion, our enzymatic extraction method yielded highly purified products while maintaining the natural properties of the SF-melanin, which could be chemically, physically, morphologically, defined as eumelanin.