PURIFICATION AND PROPERTIES OF TRANSGLUTAMINASE FROM STREPTOVERTICILLIUM MOBARAENSE

Transglutaminase (TGase) was separated from the culture broth of an isolated strain of Streptoverticillium mobaraense. The crude enzyme was prepared by centrifugation, ultrafiltration, precipitation by alcohol, centrifugation and freeze‐drying. The yield after these processes was 65–70%. Then the enzyme was purified to homogeneity by chromatography on CM‐cellulose and Sephadex G‐75 on which the yields were about 70% and 80%, respectively; the purified folds reached 2.5–4.7 and 1.08–2.06, respectively. The molecular weight of this TGase was 39,500–40,100 Da by gel filtration chromatography. Optimum enzyme activity was observed in the pH range of 5.0–7.0, and it was maintained stable at 20–40C. The optimal temperature and pH was 52C and 6.0, respectively. At 1 mM and 5 mM metal ion or inhibitors concentration, TGase activity was strongly inhibited by Zn²⁺ and NEM, and not affected obviously by Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Na⁺ as well as PMSF and EDTA. The effects of these additions on this TGase were compared with those of other microbial TGases.     

香菇谷氨酰胺转氨酶的酶学性质研究

采用超滤浓缩,乙醇沉淀,冷冻干燥和SephadexG-100层析等方法,获得香菇谷氨酰胺转氨酶粗酶及纯酶,对其酶学性质进行研究.结果表明:香菇中谷氨酰胺转氨酶粗酶的最适反应温度为40℃,温度稳定范围在50℃以下,最适PH为6.0,pH稳定范围在5.0～7.0,Na+、Ca2+等离子对该酶的活性影响甚微,为非Ca2+依赖性酶;纯酶的最适温度为40℃,以Nα-CBZ-Gin-Gly为底物时香菇谷氨酰胺转氨酶的Vmax为0.020mg/(mL·min),Km为1.520g/L.     

PURIFICATION AND CHARACTERIZATION OF A PHYTASE FROM SPELT

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K_M 400 μmoll^?1 and k_cat 368s^?1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: I. PURIFICATION AND PROPERTIES

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-100 column chromatography. The enzyme was purified 11.8 fold resulting in a specific activity of 250.3 units/mg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K_mfor catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4.11 mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and 1-naphtol did not show any activity. Data for V_max/K_m which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu²⁺ ions bound was found to be 2 per enzyme molecule.     

PURIFICATION AND CHARACTERIZATION OF A LIPASE FROM LACTOBACILLUS PLANTARUM 2739

A 65 kDa intracellular lipase from Lactobacillus plantarum 2739 was purified to homogeneity (482-fold, specific activity of 251 μmol/mg per min) and characterized. The purification procedure included chromatography with Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The purified lipase was optimally active at pH 7.5 and 35C; it retained about 40% of the maximum activity at pH 5.0 and 15C. The enzyme was stable at 65C (D_65C= 18.6 min) and was irreversibly inactivated at 75C for 2 min. On triglycerides, the highest activity was determined on tributyrin but trilaurin and tripalmitin were hydrolyzed also. The Km on tributyrin was 2.31 mM. β-Naphthyl esters of fatty acids from C2 to C12 were hydrolyzed with a preference for β-naphthyl butyrate. After lipolysis, the fatty acid profiles in β-monoacylglycerols of milk fat showed similarities among porcine pancreatic lipase, rennet paste and lipase from Lb. plantarum 2739, but the bacterial enzyme caused a greater hydrolysis of C10 and C12 fatty acids esterified at the Sn-2 position of glycerol. The lipase was strongly inhibited by 1 mM Nethylmaleimide and iodoacetic acid, by 10 mM Hg²⁺ and Ag⁺, and was moderately stimulated by Ca²⁺ and Mg²⁺.     

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean (Phaseolus mungo). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CM‐Sephadex and high performance liquid chromatography on POROS HS‐20. The purified protein exhibited a molecular mass of 38 kDa in SDS‐polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 µM. The partial N‐terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean (Glycine max) and potato (Solanum tuberosum), respectively.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A HEMAGGLUTININ FROM SEEDS OF JATROPHA CURCAS

An extract from Jatropha curcas seeds, purified by gel filtration on Sephadex G-75 and Sephacryl S-200, yielded an active hemagglutinin of high purity as assessed by electrophoresis and isoelectric focussing. The hemagglutinin had a molecular weight of around 660,000 and a pI value of 5.75. The molecule was composed of two different subunits of molecular weights 23,450 and 11,500. Amino acid analysis suggested that the molecule lacked 1/2 cystine but contained a high proportion of acidic and basic amino acids. Agglutination of trypsinized erythrocytes, groups A, B and O, took place over the range pH 4–10, and was prevented by D- galactose, D- galactosamine and N- acetyl -D- galactosamine. The hemagglutinin has only a weak binding capacity for D- galactose. Its activity was stable up to 60°C; at 80°C activity was lost in 50 min.     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

PURIFICATION AND CHARACTERIZATION OF THREEISOZYMES OF PECTIN METHYLESTERASE FROM TOMATO FRUIT

Three isozymes of pectin methylesterase (EC 3.1.1.11) have been purified to homogeneity from tomato (var. S. marzano). The isozymes were separated by affinity chromatography on Heparin-Sepharose column. They exhibited a molecular mass of 31 kDa when analyzed in sodium dodecyl sulfate gel electrophoresis and of 35 kDa in gel-filtration chromatography in native conditions. The isoelectric points of all three isozymes were found to be higher than 9.3. The K_ms calculated for the three isozymes were different toward citrus pectin used as substrate; one had a K_m of 9.7 mM (by expressing the pectin concentration as mmoles/L of methoxy groups) and the other two had similar K_ms of 3.0 and 2.6 mM, respectively. The isozyme having the higher K_m for substrate was inhibited by citrus pectin (which had a degree of methylation of 70%) at concentrations higher than 5 mM, but no inhibition was found using a pectin with a degree of methylation of 30% at concentrations up to 13 mM (i.e. 9 mg/ml) with a K_m of 14.7 mM. Furthermore, this isozyme showed a more broad range of activity in a pH range 5–10 with respect to that exhibited by the other two isozymes. All three isozymes were found to be glycosylated, although to different extents.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-POLYGALACTURONASE FROM ASPERGILLUS NIGER

A pectinase was identified and isolated from a commercial Aspergillus niger pectinase preparation. The crude enzyme preparation, which was prepared by precipitation of the water extract of the culture of A. niger with ammonium sulfate, was further fractionated by three steps of chromatography, i. e., cation exchange, hydrophobic interaction and onion exchange, to obtain an electrophoretically homogeneous pectinase. The molecular weight of the purified enzyme was estimated by SDS‐PAGE to be about 40.4 kDa under both nonreducing and reducing conditions, with the optimum pH at 5.0 and the optimum temperature at 36C. The enzyme was stable at temperatures below 35C. The partial N‐terminal ammo acid sequence data analysis of the first 19 amina acids of the obtained pectinase revealed 94.7% and 89.5% homology with two reported pectinases from A. niger.     

鲑鱼鱼精蛋白酶解产物中抗氧化活性肽的分离纯化研究

研究发现鲑鱼鱼精多肽具有较强的羟自由基、DPPH(1,1-二苯基-2-苦基肼)和超氧阴离子清除活性.通过凝胶色谱、离子交换色谱和反相高效液相色谱分离方法,最终分离到一系列具有抗氧化性的肽段.