Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

聚乙烯醇环氧化载体制备及在植酸酶固定化中的应用

该文以聚乙烯醇为原料,利用戊二醛为交联剂,聚合得到交联球形聚乙烯醇,并将其与环氧氯丙烷反应用以固载环氧基团,得到可用于酶固定化的载体。讨论了交联聚乙烯醇球环氧基的固载条件和酶的固定化条件。植酸酶经过固定化后,机械性能和化学稳定性都得到提高,可以重复多次对植酸钠进行水解反应。同时对该自由酶和固定化酶进行酶学特性研究。发现自由酶和固定化酶的最适pH值分别为5.5和7.0,最适反应温度分别为55℃和70℃。植酸酶经固定化,提高了酶的操作、温度、pH值和贮藏稳定性。     

固定化酶法生产蔗果低聚糖糖浆技术的探讨

本文探讨了固定化酶生产蔗果低聚糖糖浆的生产技术,研究证明固定化酶可生产60批次,并对生产过程中pH值、灭菌时间、灭菌温度、不同原料糖源的因素进行了探讨,对保质期内产品中蔗果低聚糖(GF2、GF3、GF4)含量的变化进行测定。     

Isolation of a new lytic enzyme for hiochi bacteria and other lactic acid bacteria

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.     

Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

Purification and characterization of a novel glutamyl aminopeptidase from chicken meat

A novel glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was purified from chicken meat by ammonium sulfate fractionation, ethanol fractionation, heat treatment, and successive column chromatographies of DEAE-Sepharose CL-6B and Sephadex G-200. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 55,000 and 550,000 by SDS-PAGE and Sephadex G-200 column chromatographies, respectively. This enzyme hydrolyzed Glu- and Asp-, but not Leu-, Arg-, and Ala-2-naphthylamide (-2NA) at all. The optimum pH and temperature for hydrolysis of Glu-2NA was 7.5. and 70°C, respectively. Reducing agents such as cysteine and dithiothreitol inhibited the activity of this enzyme at concentrations of 1 mM. However, the activation by Ca(2+) and the inhibition by amastatin were not observed.     

Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscle. I. Determination of activity in tissue extracts

It is to be expected that changes in the subcellular distribution of the mitochondrial enzymes lipoamide dehydrogenase (LIPDH), citrate synthase (CS) and beta-hydroxyacyl-CoA-dehydrogenase (HADH) in the muscle tissue give information on the type and the extent of damage of mitochondria during storage and treatment of meats; such changes may be also used as basis of methods for the differentiation between fresh and frozen/thawed meat. Standard methods for the determination of the activities of LIPDH, CS, and HADH in tissue extract and muscle press juice are described. The influence of enzyme concentration, pH and temperature on the enzyme activities in muscle extract was investigated. Furthermore the error in the enzyme analyses by the standard methods was determined.     

Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1

A novel bacterial strain capable of growing in a medium containing a medium-chain-length polyhydroxyalkanoate (MCL-PHA) as the sole carbon source was isolated from a soil sample. The isolate, which was identified as Pseudomonas sp. RY-1, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated in a medium containing a MCL-PHA, such as polyhydroxyoctanoate (PHO) or polyhydroxynonanoate (PHN). The extracellular MCL-PHA depolymerase of this organism was purified to electrophoretic homogeneity. The enzyme was a tetramer with identical subunits and a total molecular mass of 115 kDa. The isoelectric point of this enzyme was estimated to be 5.9 by isoelectric focusing. The maximal activity was observed at pH 8.5 and 35 degrees C. The enzyme was insensitive to phenylmethylsulfonyl fluoride and dithiothreitol, unlike other short-chain-length (SCL) PHA depolymerases. The K(m) values for PHO and PHN were 0.86 and 1.47 mg/ml, respectively. The enzyme could not hydrolyze SCL-PHAs and p-nitrophenyl esters of fatty acids.     

Functional display of amylase on yeast surface from Rhizopus oryzae as a novel enzyme delivery method

Fungal amylase has great importance in fermentation industry such as brewing, food fermentation, starch hydrolysis and for improving microbial populations in chicken intestine through feed applications. In the present investigation, alpha amylase cDNA from Rhizopus oryzae was cloned, sequenced, and successfully surface anchored in functional form using Saccharomyces cerevisiae EBY100 as host, yielding enzyme activity of 4.35 (±0.5) U/ml. The surface displayed yeast expressed amylase activity using plate assay, produced glucose and maltose as hydrolysis products using starch as substrate. The targeted and armed yeast with displayed enzyme was evaluated for its characterization at various pH and temperatures. The engineered yeast showed optimal activity at neutral pH and at 50°C incubation temperature. Reducing sugars produced by displayed enzyme were visualized by paper chromatography. The data suggested successful heterologous expression and display of amylase enzyme on yeast cell surface. The displayed system could further be utilized in fermentation industry for improving cost-effectiveness of the process.     

法尔皮有孢汉生酵母(Hanseniaspora valbyensis)醇乙酰基转移酶的分离纯化及特性

乙酸苯乙酯是苹果酒里的重要香气物质之一,是在醇乙酰基转移酶(AATase)的催化下由乙酰辅酶A和苯乙醇缩合而成。从法尔皮有孢汉生酵母(Hanseniasporavalbyensis)中收集细胞膜片段,然后经过DEAESepharose、SephadexG-75、OctylSepharose三步层析分离得到纯酶。纯化后的酶经SDS-PAGE电泳,得到一条明显的条带,分子量在37kD左右。此酶的最适反应条件是在30℃、pH7.0,在10℃以下酶比较稳定,pH稳定在7.0～8.0。重金属离子Hg2+、Zn2+、Pb2+对酶活有明显的抑制,EDTA、Mn2+对酶活没有太大的影响,Mg2+能激活此酶。此酶也能催化其它重要乙酸酯的合成,但对乙酸苯乙酯的合成具有特异性。     

Purification and Properties of β-Glucuronidase from the Muscle of Bombay Duck (Harpodon Nehereus)

Bombay duck muscle β-glucuronidase purified 4200-fold had molecular weight of 160,000 as estimated by gel filtration on Sephadex G-200. The glycoprotein enzyme exhibited dimeric structure on SDS-PAGE and had a pI of 5.0. The enzyme was optimally active at pH 5.2 when phenolphthalein β-D-glucuronide was used as the substrate while with p-nitrophenyl glucuronide the pH optimum was 4.6. Saccharo-1,4-lactone was a potent competitive inhibitor of the enzyme. Heavy metalic ions such as Hg²⁺, Cd²⁺ and Ag⁺ also proved to be inhibitory to the enzyme. Radiation inactivation of the enzyme could be protected in the presence of mercaptoethanol. Sodium chloride activated the enzyme while sodium tripolyphosphate inhibited it.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

The activity of myrosinase from broccoli (Brassica oleracea L. cv. Italica): influence of intrinsic and extrinsic factors

The potential of some intrinsic (MgCl2, ascorbic acid, pH) and extrinsic (temperature, pressure) factors for controlling/altering activity of myrosinase from broccoli was investigated in this paper. A combination of MgCl2 and ascorbic acid was found to enhance enzyme activity. Concentrations resulting in optimal activity were determined as 0.1 g/liter and 2 g/liter, respectively. Both in the absence and presence of this enzyme activator, the optimal pH was situated between 6.5 and 7, corresponding to the natural pH of fresh broccoli juice. At atmospheric pressure, the enzyme was optimally active at a temperature about 30 degrees C. Application of low pressure (50 to 100 MPa) slightly enhanced the activity while at higher pressure (300 MPa), the activity was largely reduced. Future work should focus on the extension of this work to real food products in order to take cellular disruption into account. In intact vegetable tissues, the enzyme myrosinase is present in compartments separated from its substrate, the glucosinolates. Hence, enzymatic hydrolysis can merely occur after cellular disruption. In this respect, processes such as cutting, cooking, freezing, or pressurizing of the vegetables will have a large effect on the glucosinolate hydrolysis by myrosinase. This work could then be the basis for controlling glucosinolate hydrolysis in food preparation and processing.     

Cyclamate intake and cyclohexylamine excretion are not related to male fertility in humans

Cyclamate and its metabolite cyclohexylamine affect male fertility in high dose animal studies, but this affect has not been investigated in epidemiological studies. This paper reports the first epidemiological study designed to investigate the possibility of a relationship between cyclamate and cyclohexylamine and male fertility in humans, in which 405 cases of clinically defined infertility in men and 379 controls were surveyed. Semen evaluation, urine analysis for cyclamate and cyclohexylamine and dietary questionnaires were compared between cases and controls. No evidence was found of a significant association between cyclamate intake and male infertility; neither high cyclamate nor high cyclohexylamine excretion were associated with elevated risk. The lack of association remained after adjusting by age, area of residence, education, total energy intake and other variables. No significant correlations were observed between cyclamate intake, metabolism or excretion, and sperm count and motility. The results demonstrate no effect of cyclamate or cyclohexylamine on male fertility at the present levels of cyclamate consumption.     

响应面法优化西兰花种子中异硫氰酸酯的酶解制备工艺

本研究采用单因素和响应面分析法优化了西兰花种子中异硫氰酸酯（Isothiocyanates，ITCs）的酶解制备工艺，分别考察了酶与底物的质量比、酶解时间、酶解温度、pH及浸提料液比对ITCs提取率的影响，在单因素实验基础上，选取酶解时间、温度及pH进行响应面试验，并使用硫脲法和气相色谱-质谱法（Gas chromatography-mass spectrometry，GC-MS）进行检测分析。结果表明，ITCs的最佳酶解条件为:酶解时间3.95 h，酶解温度44.6 ℃，pH6.52，实验提取率达到8.36 mg/g，理论提取率为8.51 mg/g，相对误差为1.8%。气相色谱-质谱测得酶解主要产物包括4-（甲硫基）丁腈、1-异硫代氰酸丁酯、萝卜硫腈、噁唑烷硫酮以及萝卜硫素五种相关化合物，其中目标活性物质萝卜硫素的相对含量最高。综上，该提取工艺参数用于西兰花种子中ITCs的制备是可行的。     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

Proteasome from rabbit skeletal muscle: Some properties and effects on muscle proteins

Rabbit proteasome, likely to be a 20S proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purified enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20000–29000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580 000–800 000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pH for hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) (Suc-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH 5–9 and up to 60 °C at pH 7.2. The enzyme activity was inhibited by diisopropyl fluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaCl₂. Remarkably, SDS increased the V_max value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, -actinin, actin, tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Purification and Characterization of an α-L-Rhamnosidase from Aspergillus terreus of Interest in Winemaking

ABSTRACT: An enzyme with α-L-rhamnosidase activity was purified to homogeneity from a culture filtrate of Aspergillus terreus after growth in a medium containing L-rhamnose as the sole carbon source. The biosynthesis of this enzyme was repressed by glucose. The enzyme had a molecular mass of 96 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 4.6 as determined by analytical isoelectric focusing. The pH and temperature optima for the enzyme were found to be 4.0 and 44 °C, respectively. Using p-nitrophenyl-α-L-rhamnopyranoside as a substrate, the enzyme exhibited Michaelis-Menten kinetics with K_M and V_max values of 0.17 mM and 84 U/mg, respectively. The enzyme was inhibited competitively by L-rhamnose (K₁ 2.5 mM). Divalent cations such as Ca²⁺ Mg²⁺ Zn²⁺ and Co²⁺ stimulated the a-L-rhamnosidase activity, whereas this was inhibited by Hg2+ and Cd2+. Ethanol (12% v/v) and glucose (21% w/v) decreased enzyme activity by approximately 20%, while this was not affected by SO₂.     

三疣梭子蟹酚氧化酶的生化特性研究

对三疣梭子蟹酚氧化酶的生化特性进行研究。结果表明：以邻苯二酚为底物,酚氧化酶在pH7.8～8.4时最稳定,最适pH 值为8.2。温度55℃时,酚氧化酶活力最大,在25～40℃时,热稳定性最好。酶促褐变反应动力学符合米氏方程,温度为30℃,pH7.2 时,米氏常数Km=0.531mol/L,最大反应速率0.049 A530nm/min。选用的12 种抑制剂对三疣梭子蟹酚氧化酶均有一定的抑制效果,以植酸抑制效果最强,焦亚硫酸钠、L- 半胱氨酸、草酸次之,抗坏血酸效果较好。