Partial Purification of Acetic Acid Esterase from Malted Barley

Partial purifications (84, 114 and 390 fold) of a soluble acetic acid esterase from barley malt that degrades diacetin have been achieved. Enzyme recoveries were 20%, 20% and 25% in three consecutive runs. Initial problems of high viscosity and colour in the extract were overcome with the use of batch‐elution anion exchange chromatography. This was followed by gradient elution anion exchange chromatography and gel filtration chromatography, which gave the greatest purification, but which gave erratic estimates of the molecular weight of the enzyme (40, 48 and 84 kDa). The apparent Km value of the acetyl esterase was tentatively estimated to be 25mM diacetin.     

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.     

Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 Strain

ABSTRACT:  For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production.     

Purification of Amylase from Honey

The major amylase in honey was concentrated by ultrafiltration, isolated by ultracentrifugation and gel filtration, and purified by ion‐exchange chromatography. The amylase activity was in the flow‐through fraction of the anion‐exchange column, suggesting a high isoelectric point (>7.4) for the enzyme. The enzyme fraction from the anion‐exchange chromatography was loaded onto a cation‐exchange column, and the amylase activity was eluted as a single band at 50 mM NaCl. The purification factor after this step was 531‐fold. The purified enzyme was an a‐amylase, as determined by thin‐layer chromatography, with a molecular weight of 57000 Da according to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The results supported the concept that amylase in honey had a high degree of similarity with bee amylase.     

Purification of an Arabinofuranosidase and Two β‐Xylopyranosidases from Germinated Wheat

Arabinosidase and β‐xylosidase activities were detected in germinated wheat grain, and both increased over seven days of germination, under malting conditions. Arabinosidase was partially purified by anion exchange chromatography, chromatofocusing and gel filtration chromatography. The pH optimum of the partially purified enzyme was 4.2 and the K_M was 1.90 mM p‐NP‐Ara. Edman degradation, MALDI‐TOF mass spectrometry and nano‐ESI mass spectrometry were used to identify the two major proteins in the partially purified arabinosidase mixture. The two proteins were a β‐amylase with an amino acid sequence partially homologous to a barley β‐amylase, and three wheat serine protease inhibitors. Further purification, by affinity chromatography and hydrophobic interaction chromatography, removed the identified contaminating proteins. At this point an 80 kDa protein was detected by SDS‐PAGE. No identity could be assigned to this protein by MALDI‐TOF mass spectrometry by reference to electronic protein databases. Similarly, the β‐xylosidase was partially purified by anion exchange chromatography followed by chromatofocusing and gel filtration chromatography. The first step separated the mixture into two distinct fractions with K_M values of 3.35 and 4.01 mM p‐NP‐Xyl and pH optima of 4.5. The latter fraction also displayed xylanase activity against RBB‐xylan.     

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein‐like activity. RESULTS: A kallikrein‐like proteinase of Agkistrodon halys pallas snake venom, designated AHP‐Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N‐glycosidase F (PNGase F). Sequence studies on the NH₂‐terminal region of the protein indicated that AHP‐Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP‐Ka showed high catalytic activity and kallikrein‐like activity on substrates such as arginine esterase BAEE and chromogenic H‐D‐Pro‐Phe‐Arg‐pNA·2HCl (S‐2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP‐Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein‐like enzyme. Copyright © 2011 Society of Chemical Industry     

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。     

Extraction and Assay of Acetic Acid Esterase From Malted Barley*

The extraction and the assay conditions for acetic acid esterase from barley malt have been optimised. An assay method was developed using diacetin as a substrate. The presence of reduced glutathione and the detergent Triton‐X‐100 in the extraction medium improved the yield of enzyme. The optimal pH for extraction was 8.0. When malt extracts were held at various temperatures acetic acid esterase was denatured at temperatures above 30°C. The optimum pH when measuring activity was 7.0. The crude enzyme extract released acetic acid from both soluble and insoluble cell wall materials. An insoluble, active form of the enzyme remained in the residual grain solids after the soluble form had been extracted.     

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.     

Use of Xylose Dehydrogenase from Trichoderma viride in an Enzymic Method for the Measurement of Pentosan in Barley

A xylose dehydrogenase has been isolated from Trichoderma viride. It has been used as part of an enzyme‐based assay to measure pentosan in barley. Pentosan is extracted with perchloric acid and then hydrolyzed by a cocktail of enzymes including xylanase, arabinofuranosidase, feruloyl esterase and acetyl esterase. Some 92% conversion to xylose was obtained, and this is measured in turn using a xylose dehydrogenase‐based assay mixture involving NAD‐dependent reduction of phenazine methosulphate monitored from absorbance increase at 585 nm. The xylose dehydrogenase is somewhat unstable and this will need to be enhanced for the assay to be functional.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

IDENTIFICATION OF THE DIMETHYL SULPHIDE PRECURSOR IN MALT

The heat-labile dimethyl sulphide (DMS) precursor in green malt has been isolated using ion exchange chromatography and gel-filtration under mild conditions. The final preparation had ～0.7 mol potential DMS per mol amino groups. Thin layer chromatography of this preparation, and the gel-filtration behaviour of both crude and purified preparations, showed that the precursor had identical properties to S-methylmethionine (SMM). The DMS precursor in a continental lager malt was also examined. The malt had been kilned under conditions which were expected to lead to a significant content of ‘active’ DMS precursor (reported to be metabolized to free DMS by suitable yeast strains, and contrasting with the ‘inactive’ green malt precursor). The DMS precursor in an extract of this kilned malt had the same elution volume on Sephadex G15 as SMM. There was no evidence for any difference from the DMS precursor in green malt. It is concluded that SMM is the only heat-labile DMS precursor in malt.     

CHARACTERISTICS OF TWO TRYPSIN ISOZYMES FROM THE VISCERA OF JAPANESE ANCHOVY (ENGRAULIS JAPONICA)

Two isozymes of trypsin (TR‐I and TR‐II) were purified from the viscera of Japanese anchovy (Engraulis japonica) by gel filtration and anion‐exchange chromatography. Final enzyme preparations were nearly homogeneous in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular weights of both enzymes were estimated to be 24,000 Da by SDS‐PAGE. The N‐terminal amino acid sequences of the TR‐I, IVGGYECQAHSQPHTVSLNS, and TR‐II, IVGGYECQPYSQPHQVSLDS, were found. Both TR‐I and TR‐II had maximal activities at around pH 8.0 and 60C for hydrolysis of Nα‐p‐tosyl‐L‐arginine methyl ester hydrochloride. The TR‐I and TR‐II were unstable at above 50C and below pH 5.0 and were stabilized by calcium ion.     

Purification and characterization of a dipeptidase from Lactobacillus delbrueckii subsp. bulgaricus

A metal-dependent dipeptidase has been purified from a cell-free extract of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography, metal chelating affinity chromatography with immobilized Cu²⁺, and repeated FPLC anion exchange chromatography. The molecular mass of the purified enzyme was estimated to be 51 kD by SDS-polyacrylamide gel electrophoresis as well as by gel filtration, which indicates that it does not consist of subunits. The enzyme was most active at pH 7 and 50°C. Reducing agents, like dithiothreitol and β-mercaptoethanol, increased enzyme activity while metal chelating agents had an inhibitory effect. Enzyme activity, inhibited by EDTA and EGTA, could be partially restored by Co²⁺ and Mn²⁺. The enzyme was most active on dipeptides containing an aminoterminal hydrophobic amino acid such as Leu-Leu and Leu-Gly. Kinetic studies indicated that the dipeptidase had a higher affinity for the first substrate mentioned. The K_m-values for both substrates were about 0·56 and 1·23 mM, with turnover numbers of 870 and 480 s⁻¹, respectively.     

PURIFICATION OF BARLET MALT α-AMYLASE BY IMMUNOAFFINITY CHROMATOGRAPHY

Immunoaffinity chromatography was used to purify the high pl α-amylase (α-amylase II) in a one step procedure after fractionation of the whole barley malt extract on Sephadex G25. The immunoglobulin G (IgG) fraction of an immune serum specific for the malt α-amylase II was immobilized on Ultrogel. A mild desorption procedure was used, combining distilled water elution with an interrupted elution. The purification was achieved within half a day including kernel extraction. The quality of the purification was assayed by SDS polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and isoelectric focusing. For the second technique, an immune serum was used which was polyspecific for malt proteins including the high pl α-amylase (α-amylase II). The effect of this procedure on the specific activity of the enzyme and on its antigenicity was evaluated. The results underline the efficiency of the purification procedure and indicate that α-amylase II accounts for a few percent of the total soluble protein in malts. However, the α-amylase II fraction was not completely free from α-amylase I. The procedure resulted in a partial loss of the enzymatic activity but not of the antigenicity.     

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。     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

Extraction of pectic polysaccharides from sugar‐beet cell walls

Previous methods of extracting pectin from sugar‐beet have used pulp as the starting material. As the temperature and pressure of the pulping process may modify the architecture of the cell wall, we have adapted a relatively non‐disruptive method to characterise cell wall material (CWM) isolated directly from the sugar‐beet. Cell walls from mature sugar‐beets (Beta vulgaris L Aztec) were sequentially extracted four times with imidazole and twice with sodium carbonate to produce six heterogeneous pectic polysaccharide extracts, and with KOH to produce a hemicellulosic extract which was predominantly xylans. Heterogeneity of the extracted pectins was indicated by differences in FTIR spectra, uronic acid content, % methyl esterification, % feruloylation, % acetylation, molecular weight distribution and neutral sugar composition. The highest proportion of feruloyl esters was found in polysaccharides solubilised by the second sodium carbonate extraction. Anion exchange chromatography of these polysaccharides gave three fractions, one of which contained most of the feruloyl ester. These results indicate that feruloyl esters are not randomly distributed among the different pectic polysaccharides in the sugar‐beet cell wall, and that esterification is likely to be dependent on the local sugar sequence or conformation. © 2000 Society of Chemical Industry     

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.     

Purification and characterization of a soluble methionyl aminopeptidase from porcine skeletal muscle

A soluble aminopeptidase was purified from porcine skeletal muscle by ammonium sulfate fractionation and two successive anion exchange chromatographic procedures. The enzyme eluted at 0.17 M NaCl, had a relative molecular mass of 53 KDa (by SDS-polyacrylamide gel electrophoresis) and was activated by sulfydryl compounds. Activity was optimal at pH 7.5 and 40°C and showed broad aminopeptidase and low endopeptidase activities. The aminopeptidase exhibited maximal activity against Met-, Lys-, Ala-, and Leu-7-amido-4-methyl-coumarin (-AMC), while Pro-AMC was not hydrolyzed. Inhibition of enzyme activity was observed in the presence of sulfydryl reagents, iodoacetic acid, puromycin, leupeptin and amastatin, but it was not affected by serin and aspartic protease inhibitors, EDTA and bestatin. The enzyme activity was not inhibited by sodium chloride and, therefore, the enzyme has potential for contributing to the generation of free amino acids in cured pork meat products.