Purification and Characterization of an Endoprotease from Melon Fruit

An endoprotease was purified from melon fruit (Cucumis melo L.) by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography using t-butyloxycarbonyl-Ala-Ala-Pro-Leu p-nitroanilide as a substrate. The molecular weight was estimated as 26,000 and isoelectric point pH 9.5. It preferentially hydrolyzed peptide bonds of the carboxyl terminal sides of Leu, Ala, His, Gin, and Am. Activity was strongly inhibited by diisopropyl phosphofluoridate, indicating the serine protease nature of the enzyme. The migration distance on electrophoresis, molecular weight and substrate specificity differed from cucumisin, a known protease from melon. This unusual protease may have potential for special food treatment applications.     

枯草芽孢杆菌内切木聚糖酶的纯化与性质研究

建立了一种快速、简便分离纯化木聚糖酶的方法。采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合 ,从枯草芽孢杆菌 (Bacillussubtilis)固态培养基发酵产物中分离得到了 2种内切木聚糖酶 ,分别定义为xylⅠ和xylⅡ ,它们水解桦木木聚糖的主要产物有木二糖、木三糖和聚合度更高的木聚寡糖 ,没有木糖。SDS PAGE显示内切木聚糖酶xylⅡ为单肽链结构 ,分子质量为 98 8ku。内切木聚糖酶xylⅡ的酶反应最适温度为 5 0℃ ,酶反应的最适 pH为 7 0。Mn2 + 对xylⅡ酶反应具有促进作用 ,将酶活提高了 2 7倍 ,而Fe3+ 对该酶反应起完全抑制作用。     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM HEPATOPANCREAS OF CRAWFISH (PROCAMBARVS CLARKII)1

Four trypsin‐like enzymes (CP‐I, II, III and IV in order of elution on DEAE‐Sepharose chromatography), purified from the hepatopancreas of crawfish, were inhibited by protease inhibitors such as phenyl methyl suifonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), aprotinin and tosyl lysine chloromethyl ketone (TICK). The molecular weights of CP‐I, II, III and IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacryl‐amide gel electrophoresis (SDS‐PAGE), These proteases had optimal esterase activity at pH 8.0–8.5 and showed the highest activity at 60–70C. Crawfish proteases were rich in acidic amino acids. Activation energies for hydrolysis of tosyl arginine methyl ester (TAME) by these proteases were 6.98 – 8.34 kcal/mole. Unlike other serine proteases, the activities of CP‐I and CP‐II were activated by mercury while CP‐HI and IV were inhibited.     

Purification and Characterization of a Trypsin‐Like Protease from Flatfish (Paralichthys olivaceus) Intestine

A trypsin‐like protease was purified from the intestine of flatfish (Paralichthys olivaceus) by gel filtration and anion‐exchange chromatography. The molecular weight was estimated to be 29.6 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Flatfish protease had maximal activity at 70C and pH 7.5 using N‐α‐benzoyl‐dl ‐arginine‐ρ‐nitroanilide as substrate. It was stable to heat treatment up to 50C and to pH ranges between 7.0 and 10.0. It was activated by calcium ion and completely inhibited by mercury ion and known serine‐protease inhibitors, such as phenylmethylsulfonyl fluoride, tosyl lysine chloromethyl ketone and benzamidine.     

Partial purification and characterization of proteases from Norway lobster (Nephrops norvegicus) and their role in the phenolase activation process

Proteolytic activity in Norway lobster (Nephrops norvegicus) was studied. An improved separation and partial purification of the three proteases (designated as proteases I, II and III) was achieved from Norway lobster heads by a combination of acetone precipitation and DEAE-Sepharose CL-6B column chromatography.The purification achieved was 63-, 25- and 217-fold at pH 8.2, and 40-, 25- and 160-fold at pH 6.4 for protease I, II and III, respectively.With casein as substrate, protease III was most active at pH 8.2, whilst proteases I and II showed activity over a wide range of pH.Protease III was characterized as an alkaline Zn-serine protease as it was strongly inhibited by PMSF, soybean trypsin inhibitor, Co²⁺, Mn²⁺ and 1–10 phenanthroline. Protease I was strongly inhibited by p-benzoquinone, iodo-acetamide, heavy metals (Ag⁺, Cu²⁺) and 1–10 phenanthroline and was thus characterized as a Zn-thiol protease. Protease II was also inhibited by the same inhibitors as protease I (but to a lesser extent) and was characterized as a thiol protease.The molecular weights were determined to be 22.5, 45 and 42.5 kDa (with activity at 18 kDa) for proteases I, II and III, respectively.It was found that protease III activates phenolase at pH 8.2, whilst proteases II and I can activate phenolase at both pH 6.7 and 8.2.     

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.     

Hydrolysis of Milk Protein by a Bacillus licheniformis Protease Specific for Acidic Amino Acid Residues

A serine protease, which preferentially cleaves peptide bonds at the carboxylic site of Glu and Asp was evaluated with milk proteins as substrate. The enzyme hydrolyzed casein almost 10 times more efficiently than whey protein. In the casein assay, whey protein did not inhibit the protease, but the enzyme activity in a chromogenic assay was severely inhibited by one whey protein, β-lactoglobulin. Capillary electrophoresis of β-lactoglobulin hydrolysate revealed a peptide profile corresponding to the numbers of susceptible bonds, The enzyme may provide advantages in preparation of functional protein fractions and in cheese ripening.     

响应面法优化大黄花鱼肉蛋白水解工艺及多肽抗氧化性研究

以大黄花鱼为实验材料,利用酶法水解大黄花鱼肉蛋白制备抗氧化肽。以还原力为响应值,通过单因素结合响应面法对中性蛋白酶酶解大黄花鱼肉蛋白的酶用量、酶解温度、底物浓度以及酶解时间进行了优化,结果表明:四种酶中,中性蛋白酶酶解的酶解液水解度(DH)和还原能力最高。最优酶解工艺条件为酶用量为0.4%、酶解温度45 ℃、底物浓度25.0%、酶解时间7 h、体系pH7.0时,还原力为0.951。酶解液DH为37.51%,超氧阴离子自由基清除力(O₂-·)为82.42%。SDS-PAGE(聚丙烯酰氨凝胶电泳)结果显示,酶解7 h大黄花鱼肉蛋白肌动蛋白完全消失,水解形成肌球蛋白轻链分子量为27、15和6 kDa。     

Purification and characterization of heat-stable proteases from Bacillus stearothermophilus RM-67

The extracellular proteases of Bacillus stearothermophilus RM-67 were purified by ammonium sulfate fractionation (40 to 70% saturation), gel filtration through Sephadex G-100, and diethylaminoethyl-Sephadex A-50 ion-exchange chromatography. Gel filtration resulted in separation of the enzyme preparation into one minor (protease I) and one major (protease II) peak. The three-step purification scheme resulted in 39.5-fold purification and an overall recovery of 8.1% of protease I and 87.8-fold purification and 59.7% recovery of protease II. Purified proteases had pH and temperature optima of 8.0 and 70 degrees C. Protease I and II, when together, retained 100% activity at 60 degrees C for 30 min. Manganese imported 100% stability to the pooled proteases at 65 degrees C for 30 min. Amino acid analysis of the major peak (protease II) revealed the absence of half cystine and methionine. Protease I and II had molecular weights of 67,610 and 19,950 and Michaelis-Menten constants (casein) of 1.33 and 2.0 mg/ml. Energy of activation was 14,300 cal/mol for protease I and 11,150 cal/mol for protease II. Corresponding heat of activation was l3,620 and 10,470 cal/mol.     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM OYSTER (CRASSOTREA GIGAS)

Proteases in oyster (Crassotrea gigas) were extracted with 10 mM Tris-HCl buffer solution and purified by ammonium sulfate fractionation, Sephadex G-150 gel filtration, repeated DEAE-Sephadex A-50 and CM-Sepharose CL-6B chromatography. Three fractions with caseinolytic activity, named I, II and III, were obtained from CM-Sepharose CL-6B and DEAE-Sephadex A-50 chromatography. The three proteases were purified to electrophoretic homogeneity. Substrate specificity studies indicated that protease I was a carboxypeptidase A-like enzyme; II and III were trypsin-like enzymes. The optimal pH of protease I for hydrolysis of hippuryl-L-phenylalanine was 9.0, II and III for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester (TAME) was 8.0. The temperatures which inactivated 50% of enzymes were 78°C for protease I in 30 min; 50 and 52°C for protease II and III, respectively, in 5 min. The molecular weights of proteases I, II and III were 23,000, 34, 400 and 31, 000, respectively.     

PURIFICATION AND PROPERTIES OF TRYPSIN-LIKE ENZYMES AND A CARBOXYPEPTIDASE A FROM EUPHAUSIA SUPERBA

ABSTRACT Five proteases designated as A_1, A_2, B, C and D were isolated from Euphausia superba by the succesive steps of ammonium sulfate fractionation, acetone precipitation, gel filtration and DEAE-Sephadex A-50 chromatography, A_1, B, C and D were purified to homogeneity in disc gel electrophoresis by means of rechromatography on the DEAE-Sephadex A-50 column. Studies on substrate specificity of these enzymes revealed that A_2, B, C and D were trypsin-like enzymes and A₁ a carboxypeptidase A. A_1, B, C and D had molecular weights of about 24,000, 24,000 28,000 and 27,000; optimal pH at 9.0, 8.0, 7.5 and 7.5–8. 0; and optimal temperature at 48, 50, 55–60 and 55°C, respectively. The activity of B, C and D were not inhibited by sulfhydryl reagents and N-α-tosyl-L-phenylalanylchloro-methyl ketone, but inhibited by reducing agents, N-α-tosyl-L-lysylchloromethyl ketone and soybean trypsin inhibitor. The activity of protease A₁ was stimulated 3.5 fold by cobalt, but inhibited by 3-indolepropionate and D-phenylalanine.     

Protease Activities in Raw Milk Determined Using a Synthetic Heptapeptide Substrate

A synthetic heptapeptide (H-Pro-Thr-Glu-Phe-[p-nitro-Phe]-Arg-Leu-OH) was used as substrate for detection and assay of cathepsin D in raw bovine milk. Cathepsin D produced a specific peptide, as detected by HPLC analysis of peaks for product and substrate. On incubation of acid wheys from milk samples with the substrate, three hydrolysis products were detected and bonds cleaved were identified by mass spectrometry. Inhibition studies were performed to identify enzymes responsible for the hydrolysis. One activity was cathepsin D and production of another peptide was inhibited by cysteine protease inhibitors, suggesting cysteine protease activity in milk.     

酪蛋白源降胆固醇肽的酶解制备工艺优化

以酪蛋白为原料,采用中性蛋白酶、碱性蛋白酶以及胰蛋白酶对酪蛋白进行水解,确定制备降胆固醇肽的最佳蛋白酶;通过单因素实验和响应面试验,研究水解pH、水解温度、酶与底物比、底物浓度和水解时间对酪蛋白水解度和胆固醇胶束溶解度抑制率的影响,确定最佳水解条件;而后通过超滤和凝胶过滤层析确定降胆固醇肽的初步分离工艺。结果表明:制备酪蛋白源降胆固醇肽的最佳水解工具酶是中性蛋白酶,其最佳酶解条件为反应温度51.3 ℃,酶与底物浓度比6.47%,pH6.34,底物浓度5 g/100 mL,反应时间3.5 h,胆固醇抑制率为58.25%±0.59%;Sephadex G-10分离酪蛋白降胆固醇肽条件为上样浓度80 mg/mL,上样体积2.5 mL,洗脱速度3.5 mL/min;经酶解、超滤及层析后制备的酪蛋白源降胆固醇肽峰1和峰2样品在100 μg/mL的胆固醇溶解度抑制率为24.2%±0.24%和4.3%±0.16%。经酶解制备分离后,获得具有抑制降固醇胶束溶解活性的降胆固醇肽,为降胆固醇肽的开发提供理论研究基础。     

Isolation and characterization of a protease from the <Emphasis Type="Italic">Actinidia arguta</Emphasis> fruit for improving meat tenderness

An protease from Actinidia arguta for improving meat tenderness was purified, characterized from wild A. arguta fruit by ammonium sulfate precipitation, Sephdex G-25 gel filtration chromatography, and DEAE Sepharose Fast Flow ion exchange chromatography, and its activity was investigated. The purified protease was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to obtain a single band of protease. The protease was purified successfully, and found to have a molecular weight of 23.8 kDa (mass spectrometry). The specific activity of the purified protease reached 53,428 U/mg with a 25.5-fold purification factor and 9% activity recovery. Based on N-terminal sequencing results, the A. arguta protease was derived from the class of actinidia proteases that have an N-terminal sequence of VLPDY VDWRS AGAVV. The protease was effective for tenderizing beef and decomposing actomyosin, suggesting the potential application for improving meat tenderness.     

PURIFICATION AND CHARACTERIZATION OF TWO TOMATO POLYGALACTURONASEISOENZYMES

The properties of tomato polygalacturonases at two ripening stages were investigated. Two isoenzymes, PG I and II, were isolated from underripe fruits with an orange skin color. Fully ripe fruits contained only polygalacturonase II. PG I and II were purified by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and CM-agarose chromatography. PG I had a M_r of 199,500 as determined by Sephacryl S-300 gel filtration and was 50% inactivated at 66.5°C and pH 4.5 after incubation for 5 min. It had an activation energy (E_a) of 16.8 Kcallmol (70.3 times 10³ Jlmol), V_max of 27.7 units/mg protein and K_m value of 7.5 times 10⁻² mM polygalacturonic acid. PG II had a M_r of 45,700 and was 50% inactivated at 58°C under the same conditions. Both isozymes had a pH optimum of 4.6. PG II had an E_a value of 14.8 Kcallmol (61.9 times 10³ Jlmol), V_max value of 58.8 units/mg protein and K_m value of 3.8 times 10⁻² mM polygalacturonic acid. PGI gave rise to only one band during electrophoresis in polyacrylamide gels, whereas PG II showed one major and one minor band both with PG activity. Gel electrophoresis in the presence of sodium dodecyl sulfate resulted in two major bands (M_r= 47,500 and 41,000) for PG I and only one major band (M_r= 47,500) for PG II. PG I is composed of several subunits, all of which are glycoproteins.     

长牡蛎肉小肽营养液的研制

研究长牡蛎肉小肽营养液制备的工艺条件。先以水解度为指标,确定长牡蛎肉中性蛋白酶、木瓜蛋白酶、菠萝蛋白酶单酶的最适水解条件,通过正交试验确定各单酶水解的最佳工艺条件;然后以此为基础进行三酶复合试验,根据水解度确定最佳工艺条件,制备长牡蛎肉水解液;最后以该水解液为原料,通过正交试验确定最佳配方,按最佳配方制备小肽营养液。在最佳工艺条件下,长牡蛎肉三酶复合水解度达52.97%,经聚丙烯酰胺凝胶垂直板电泳测定,小肽峰值相对分子质量范围在300～1 000之间;17种游离氨基酸含量500.16 mg/100 mL;小肽营养液最佳配方:水解液(mL)∶蔗糖(g)∶柠檬酸(g)∶氯化钠(g)为100∶10∶0.2∶0.2,游离氨基酸总量≥5 000mg/L。本小肽营养液营养丰富,制备工艺较简单,可应用于长牡蛎肉的高值化开发。     

PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM PYLORIC CAECA OF BIGEYE SNAPPER (PRICANTHUS MACRACANTHUS)

Trypsin from the pyloric caeca of bigeye snapper was purified and characterized. Trypsin had an apparent molecular weight of 23.8 kDa when analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and substrate‐gel electrophoresis. The trypsin fraction consisted of three isoforms as evidenced by the appearance of three different bands on native‐PAGE. Optimal activity was observed at 55C and pH range of 8–11. The activity of trypsin fraction was completely inhibited by soybean trypsin inhibitor and was partially inhibited by E‐64 and ethylenediaminetetraacetic acid. CaCl₂ partially protected the trypsin fraction from activity loss at 40C, while NaCl (0–20%) decreased the activity in a concentration‐dependent manner. The apparent Michaelis–Menten constant (K_m) and catalytic constant (k_cat) were 0.312 mM and 1.06 s, respectively when Nα‐Benzoyl‐dl ‐arginine ρ‐nitroanilide was used as a substrate. Trypsin from the pyloric caeca of bigeye snapper generally showed similar characteristics to other fish trypsins.     

裸燕麦球蛋白酶解液的纯化及其抗氧化研究

通过Osborne法制得的裸燕麦球蛋白,利用碱性蛋白酶对其进行酶解,采用Sephadex G-25凝胶层析对酶解液进行纯化及其分子质量测定,然后对各分离组分清除 ·OH、O2－ ·、DPPH自由基能力进行研究。结果表明：酶解液经分离纯化得到图谱为2个峰,分别为组分I(分子质量10738～133929D)和组分II(分子质量114～861D)。测得组分II清除 ·OH(IC50 0.589mg/mL)、O2－ ·(IC50 1.783mg/mL)、DPPH自由基(IC50 0.095mg/mL)能力高于组分I和酶解液。     

PURIFICATION AND CHARACTERIZATION OF INVERTASE FROM DATES (PHOENIX DACTYLIFERA L., VAR. ZAHDI)

Zahdi dates (Phoenix dactylifera) contain invertase at all development stages; the highest specific activity is present in the late yellow stage. The enzyme was purified to homogeneity, as determined by disc gel electrophoresis and isoelectric focusing, by a combination of techniques including ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sepharose 4B and Sephadex G-150 columns. A complex of invertase with a high molecular weight pectic substance of the date could not be dissociated by ammonium sulfate or DEAE-cellulose chromatography but the complex was dissociated by gel filtration on a Sepharose 4B column at pH 4.0 and ionic strength of 0.5 M. The enzyme contained 8.2% carbohydrate covalently linked probably via an amide linkage to aspartic acid. Molecular weight determination by exclusion gel chromatography and sedimentation equilibrium gave values of 130,000 and 97,100 ± 1,300, respectively. The enzyme is probably composed of two identical subunits as shown by SDS polyacrylamide gel electrophoresis. Amino acid analyses showed the enzyme to be low in sulfur-containing amino acids. Date invertase is an acid β-fructofuranosidase with a pH optimum between 3–4 and with a K_m and k_cat for sucrose of 6mM and 49 sec^-1, respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 48.7 and 17.6 kcal/mole, respectively. Chemical modification indicated that sulfhydryl groups are probably not essential for activity while carboxyl groups may be involved in the active site of the enzyme.