Extraction and Assay of Ferulic Acid Esterase From Malted Barley*

Ferulic acid esterase activity was detected in extracts of barley malt using an assay employing a novel artificial substrate, mono‐feruloyl glycerol. Mono‐feruloyl glycerol has been synthesized and analysed to determine its degree of substitution and purity. It consists of a mixture of the two isomers 1‐feruloyl glycerol and 2‐feruloyl glycerol. The extraction of ferulic acid esterase and its assay conditions have been optimised. The presence of both a detergent and reduced glutathione in the extraction medium increased the amount of enzyme extracted. A pH of 7.5 was optimal for enzyme activity. The enzyme in solution was only stable up to 30°C. The crude extract containing the enzyme released free ferulic acid from both soluble and insoluble cell wall materials. After extraction of the soluble enzyme, insoluble enzyme, capable of releasing free ferulic acid from feruloyl glycerol, was detected in the residual grain solids.     

小麦与小麦芽阿魏酸酯酶酶活力的测定及其酶学性质研究

以阿魏酸乙酯作为底物,确定了阿魏酸酯酶的酶解工艺条件:酶解时间100 min;酶液与底物的体积比为0.75;提取液pH值6.2。针对阿魏酸酯酶酶学性质的研究表明:最适温度60℃,30～35℃范围内热稳定性较强,保温60 min后其活力可保持在80%以上;最适反应pH值5.0,在pH值5.0～5.6范围内对阿魏酸酯酶的破坏力相对较小,保温60 min其活力仍可保持在80%以上;Cu2+和Zn2+对阿魏酸酯酶有强烈的抑制作用,而ED-TA则对其有明显的促进作用。对5种小麦及其麦芽的阿魏酸酯酶酶活力进行测定:麦芽中的阿魏酸酯酶酶活均高于小麦,其中以烟2415、鲁麦21、郑麦004变化较大。     

Storage Effects on Lipase Activity in Fresh-cut Cantaloupe Melon

ABSTRACT: Lipase enzyme in fresh-cut cantaloupe melon ( Cucumis melo L. var. reticulatus Naud) was assayed, and the effects of storage at 4 and 15 °C on enzymatic activity were determined. The enzyme was stable for 24 h at both temperatures but decreased by 45% after 120 h of storage time at 15 °C. Isoelectric studies suggest a possible dual lipase and esterase action of the enzyme. Enzymatic activity increased by about 60% when the reaction temperature was increased from 30 °C to 40 °C and remained constant until 70 °C, but the enzyme was unstable when stored at the high temperature. Calcium treatment reduced lipase activity in the fresh-cut fruit.     

Extracellular protease from Mucor pusillus: purification and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl₂ concentration of 0.02  m and at milk pH of 5. These properties, except for temperature, were similar to those of rennet.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

A heat-stable trypsin from the hepatopancreas of the cuttlefish (Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins.     

深绿木霉嗜酸性阿魏酸酯酶酶学性质及生物质转化分析

为实现生物质的高效降解,制备具有重要生理功能的阿魏酸,本实验从深绿木霉XS6发酵液中分离纯化深绿木霉阿魏酸酯酶（Trichoderma atroatroviride feruloyl esterase,TaFAE）,并研究酶学特性。发酵液经(NH4)2SO4沉淀、DEAE-Cellulose DE52阴离子交换层析和Sephadex G-200凝胶过滤层析后,得到电泳纯的TaFAE,比活力134.3 U/mg,回收率28.9%,纯化倍数31.52。经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得酶分子质量约为66.4 kDa。TaFAE对阿魏酸甲酯亲和力最高,以其为底物,酶Km值和Vmax值分别为0.53 mmol/L和16.74 μmol/（min·mg）。以芥子酸甲酯为底物,酶促反应速率最大,达到32.21 μmol/（min·mg）,是阿魏酸甲酯的2 倍,对咖啡酸甲酯无活性,说明该酶具有严格的底物特异性。TaFAE最适pH值和温度分别为pH 4.0和40 ℃。在pH 2.0～9.0的范围内,30～40 ℃温度下都表现出良好的稳定性。金属离子Ca2＋和Mg2＋对TaFAE活性有显著激活作用,重金属离子Hg2＋和Pb2＋几乎完全抑制酶活性;β-巯基乙醇、二硫苏糖醇、十二烷基硫酸钠、Trition X-100增强酶活性,苯甲基磺酰氟有较强的抑制作用。木聚糖酶降解生物质的体系中加入TaFAE可显著增加阿魏酸和还原糖产量,表明TaFAE和木聚糖酶有良好协同作用。综上所述,TaFAE优良的耐酸性及其他酶学性质说明其在食品和饲料行业有较好的应用潜力。     

PARTIAL PURIFICATION AND CHARACTERIZATION OF α-GALACTOSIDASE FROM ASPERGILLUS ORYZAE

A crude extract of α-galactosidase obtained by fermenting Aspergillus oryzae on wheat bran was purified 35 fold by ethanol precipitation, gel filtration and ion-exchange chromatography. The final preparation was free of protease activity but contained invertase activity. The molecular weight of the enzyme was estimated as 64,000 daltons. The pH and temperature optima were 4.0 and 60°C, respectively. The enzyme was stable over the pH range 3–7.5 and at temperatures up to 55°C (pH 4.0). The K_m values for p-nitrophenyl α-Dgalactopyranoside (PNPG) and raffinose were 4.0 × 10⁻⁴M and 1 × 10⁻²M, respectively. Divalent cations were not required for activity. More than 80% of the oligosaccharides in soy milk were hydrolyzed after 3 h at 50°C using 0.113 PNPG units/mL milk.     

Purification and characterisation of an alkaliphilic esterase from a culinary medicinal mushroom, Sparassis crispa

In this study, we found that the fruiting body of the medicinal and edible mushroom Sparassis crispa produces an alkaliphilic esterase. The substrate specificity of this esterase was high for a p-nitrophenyl acetate substrate. The S. crispa esterase was purified using ammonium sulphate precipitation, anion exchange and gel filtration chromatography. The recovery and purification yields of the enzyme were 15–17% and 70–73 folds from six different strains of S. crispa, respectively. The molecular weight of the purified enzyme was approximately 60 kDa, as determined by SDS–PAGE. A zymogram analysis using a tributyrin substrate revealed that this enzyme is an esterase. The optimum pH and temperature were 8.0 and 50 °C, respectively. The pH and temperature stability profiles show that this enzyme is more stable under alkaline conditions and at 30–40 °C. K_m and V_max for this esterase enzyme acting on p-nitrophenyl acetate were 0.2 mM and 0.5 U/mg proteins, respectively.     

Esterolytic and Lipolytic Activities of Lactobacillus Casei-subsp-Casei LLG

The estcrolytic and lipolytic enzymes were produced by cell lysis of Lactobacillus casei-subsp-casei LLG during the late logarithmic growth phase. The enzyme was purified to 67 fold by ion exchange chromatography and gel filtration chromatography using the FPLC system. Polyacrylamide gel electrophoresis and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis using the “Phast” system of the purified enzyme showed a single protein band for butyrate-esterase (3.2 × 10⁵ Dalton), caproate esterase (1.1 × 10⁵ Dalton) and capryate esterase (4.0 × 10⁴ Dalton), respectively. The maximum lipolytic activity was observed at pH 7.2 and 37°C. The enzyme activity was inhibited by silver and mercury ions but magnesium and calcium stimulated lipolytic activity. The Km and Vmax values for esterase-lipase of the strain LLG were 76 μM/min/mg of protein, and 0.57 mM, respectively. This enzyme was stable at room temperature for at least 2 days.     

CHARACTERIZATION OF AFFINITY-PURIFIED TRYPSIN FROM HYBRID TILAPIA (TILAPIA NILOTICA/AUREA)

Trypsin was extracted from the intestines of tilapia and purified 136-fold on the basis of amidase activity by a sequence of ammonium sulfate fractionation, acetone precipitation and affinity chromatography on soybean trypsin inhibitor bound to 4.0% beaded agarose. The purified trypsin exhibited higher esterase than amidase activity at its optimum pH of 9.0. The enzyme was stable in the range of pH 7–9. The optimum temperature for enzyme activity was 40C and the Q₁₀ for enzyme activity was 1.7 from 20C–30C and 30C–40C. The Arrhenius energy of activation was 8.9 and 8.7 kcal./mol respectively, for the ranges of 20C–30C and 30C–40C.     

阿魏酸酯酶的制备

阿魏酸酯酶是一种新型酶制剂,在食品、饲料和造纸工业中都有着广阔的应用前景,但目前还没有工业化产品。文中研究了超滤和冷冻干燥制备阿魏酸酯酶的工艺条件,结果表明,酶液pH值为8.06时有利于酶蛋白截留;装液厚度为6mm时,酶粉剂冷冻干燥时间为24h;添加1%～2%的聚乙二醇可以保护酶液在冷冻干燥过程中不失活;阿魏酸酯酶的液体和固体产品在保存过程中均表现出了较好的稳定性。     

Partial Purification of Ferulic Acid Esterase from Malted Barley

A partial purification of ferulic acid esterase, which degrades feruloyl glycerol, has been achieved from barley malt in small yields. Coloured and viscous materials were removed from the malt extract using batch‐elution anion exchange chromatography. Further steps included gradient elution anion exchange chromatography and gel filtration chromatography. Estimations of the molecular weight varied greatly from 22KDa to 158 KDa, possibly because the protein interacted with the matrix of the gel exclusion chromatography column and because multiple forms of the enzyme were present. The partially purified feruloyl esterase had an apparent Km of 0.46% feruloyl glycerol. However more than one enzyme may be present and the substrate contains two isomers and so Michelis‐Menten kinetics may not be appropriate.     

Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

New carboxylesterase from organic-solvent-tolerant Bacillus licheniformis S-86 strain was characterized. The enzyme named as type II esterase showed an optimal activity in the temperature range 60–65 °C and pH 8.0. The enzyme was moderatly thermostable (half-life of 1 h at 50 °C), but remarkable stable at extremely alkaline pH, retaining 100% of its activity at pH 10.0–11.0. Furthermore, the esterase showed high stability in detergents (86% residual activity in 10% SDS), and also 0.1% ionic and non-ionic detergents are inducers of enzyme activity. PMSF, a serine protease inhibitor, did not show any effect on the activity. The immobilized type II esterase was able to synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate (acyl donor) in n-hexane. The resulting ester yield (42.8%), obtained at a low temperature (28 °C) and with a very low amount of enzyme (4.6 × 10^?5 mg ml^?1), indicates a high potential for type II esterase in isoamyl acetate synthesis for production purposes.     

Partial characterization of the in situ activity of pectinesterase in Bramley apple

The characteristics of pectinesterase (PE) have been examined in waste material (peel, cores and offcuts) obtained from Bramley seedling apples barn stored for 2, 4 and 12 weeks. A crude enzyme extract was prepared by suspending the dried and milled apple waste in 0.1 m NaCl at pH 8.5. The activity of PE under standard assay conditions of 0.5% apple pectin, 0.1 m NaCl, pH 8.5 and 30°C was low, between 4 and 8 units g⁻¹ dry matter, but activities up to 60 units g⁻¹ dry matter were obtained at higher temperatures. The optimum temperature was 60°C with the enzyme stable up to 40°C with 5 min heating. The mean activation energy for PE in the three samples was calculated at 39.2 kJ mol⁻¹ K⁻¹. The optimum pH was high at 10.0 probably due to the PE assay measuring the extraction/solubilization and stability of the enzyme in addition to its activity. Optimum activity was obtained in 0.15 m NaCl with optimum stability at 0.5 m.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Rennin-like milk coagulant enzyme produced by a local isolate of Mucor

Among 20 isolates of Mucor isolated from various environments in Jordan and found to produce a rennin-like acid protease, known as Mucor rennin-like enzyme (MRE), Mucor J20 was found to produce the highest level of MRE. The optimum incubation conditions for enzyme production in a fortified wheat bran mixture using solid-state fermentation were 3–4 days at 30°C. The highest MRE activity (185–200 rennin units or RU) was produced in a medium containing wheat bran and lentil straw (1 : 1 w/w) moistened with whey, and incubated in clay pots at 30°C for 4 days. A slightly lower activity value (178 RU) was found when using a mineral salt solution or distilled water instead of whey, or when using wheat bran alone with whey. At pH 4, the MRE retained its complete activity (100%) for 6 weeks at 5°C and 10°C, and for 3 and 2 weeks at 20°C and 30°C, respectively. After heating at 60°C for 10 min, the enzyme lost its activity at all pH levels used (pH 2–8). The crude extract of MRE was successfully applied in the manufacture of a cheese curd.     

Chromatographic Isolation and Characterization of a Novel Peroxidase from Large Lima Legumes

ABSTRACT:  A novel peroxidase with antifungal activity was isolated from the large lima bean ( Phaseolus limensis ) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and gel filtration on Sephadex G-75. The enzyme was adsorbed on Affi-gel blue gel and SP-Toyopearl, and possessed a molecular weight of 34 kDa in SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. There was an almost 110-fold increase in specific activity of the purified peroxidase compared with that of the crude extract. The enzyme exhibited a pI of 8.6 by isoelectric focusing electrophoresis, indicating that it is a basic protein. The optimum pH and the optimum temperature were 5.5 and 30 °C, respectively. The enzyme was stable up to 55 °C. It potently suppressed mycelial growth in Fusarium solani, Mycosphaerella arachidicola, and Pythium aphanidermatum with an IC₅₀ of 76, 103, and 119 μM, respectively.     

ACTIVITY AND PRIMARY CHARACTERIZATION OF ENZYME FROM THERMUS RUBER CELLS CATALYZING CONVERSION OF MALTOSE INTO TREHALOSE

Thermophilic bacteria Thermus ruber produces enzyme, which catalyzes the conversion of maltose into trehalose. The specific activity of the cell-free extract from this bacteria growing without inducers was 0.028 U/mg protein and it was increased to up to 0.086 U/mg in the presence of 0.5% of maltose in the culture broth. The maximum degree of maltose conversion of about 90% was attained at 10% substrate concentration. The enzyme from Thermus ruber does not catalyze formation of trehalose from maltotetraose and maltopentaose. The optimum temperature for the enzyme activity was 65C. A maximum activity of the maltose conversion was performed at pH 6.5. The highest enzyme activity was achieved during cell cultivation at 55C on a media composed from 0.5% of peptone, 0.1% yeast extract and 0.5% of maltose or starch.

PRACTICAL APPLICATIONS

Trehalose is a chemically stable nonreducing disaccharide which can be used in food, cosmetics, medical and biotechnological industries. Extraction of this carbohydrate from yeast cells or other natural sources is unsuitable for trehalose production because of low process yield and high cost. Thus, the enzymatic methods of trehalose production are developed. In the current study the thermophilic bacteria Thermus ruber has been examined as a new source of the enzyme catalyzing conversion of maltose into trehalose.     

Gene cloning and characterization of a trehalose synthase from Corynebacterium glutamicum ATCC13032

Trehalose synthase (TreS) is an enzyme which produces trehalose from maltose through intramolecular transglycosylation. In this study, a gene (cg2529) encoding for TreS from Corynebacterium glutamicum (CgTS) was cloned and expressed in Escherichia coli. The hexahistidinetagged CgTS showed an optimum temperature and pH of 35°C and pH 7.0, respectively. This enzyme was not thermostable, but stable in a broad pH range from pH 5.0 to 8.5. Its activity slightly increased by 5 mM Mg²⁺ and Fe²⁺, while it was strongly inhibited by 5 mM sodium dodecyl sulfate (SDS). CgTS catalyzed the conversion from maltose into trehalose, and vice versa. Lowering reaction temperature by 5°C from the optimum temperature significantly reduced hydrolysis activity to produce glucose as a by-product compared to transglycosylation activity to produce trehalose, leading to increase in the conversion yields from maltose into trehalose. Consequently, the maximum conversion yield by CgTS reached 69% at 25°C after 9 hr of reaction.