酶法水解梅鱼蛋白的实验研究

主要研究了碱性蛋白酶、风味蛋白酶、复合蛋白酶、中性蛋白酶对小梅鱼的水解作用。采用正交试验,详细比较了温度、酶浓度、水解时间、酶比例对鱼蛋白水解度的影响。结果表明:单酶最佳水解酶为碱性蛋白酶,其水解条件:温度60℃,加酶量0.3%(以蛋白含量计),时间5h,水解度达51.02%;碱性蛋白酶与复合蛋白酶双酶的最佳水解条件:碱性蛋白酶与复合蛋白酶比例为3∶1,温度为60℃,加酶量0.3%,时间5h,水解度达57.03%;碱性蛋白酶、复合蛋白酶与风味蛋白酶三酶的最佳水解条件:碱性蛋白酶与复合蛋白酶比例为3∶1,温度60℃,加酶量0.3%,反应5h后添加风味蛋白酶,其反应条件:加酶量0.3%,温度60℃,时间为6h,水解度达62.57%。     

水酶法提取花生水解蛋白的研究

采用水酶法从冷榨花生饼中提取水解蛋白。以溶出的蛋白质浓度为指标,研究了提取花生蛋白的酶解工艺及条件。结果表明,先用碱性蛋白酶水解再用中性蛋白酶水解效果优于单一酶,碱性蛋白酶最适条件为：温度为55℃,pH值为9．0,加酶量为0．8％,水解时间为3h;中性蛋白酶最适条件为：温度为50℃,pH值为6．5,加酶量为0．3％,水解时间为1．5h。此条件下,蛋白提取率可达90．29％。     

金鲳鱼内脏酸性蛋白酶的分离纯化及酶学性质研究

以金鲳鱼内脏为原料,依次采用p H7.0的磷酸钠缓冲液浸提,0~55%硫酸铵沉淀,Sephadex G-100和Sephadex G-50凝胶过滤法得到纯化的酸性蛋白酶,并研究了其酶学性质。结果表明,纯化酶的分子量为18.5 ku,最适p H为4.0,最适温度为35℃,具有较好的酸稳定性和耐盐能力;以血红蛋白为底物时,该蛋白酶的米氏常数Km为10.13 g/L;胃蛋白酶抑制剂(pepstatin A)对该酶活性有抑制作用,而乙二胺四乙酸(EDTA)、反-环氧丁二酰基-L-亮氨酰胺基(4-胍基)丁烷(E-64)和苯甲基磺酰氟(PMSF)对此酶活性基本无影响,据此推断该蛋白酶是一种天冬氨酸蛋白酶;此酶对罗非鱼鱼皮明胶的酶解能力强于猪胃蛋白酶,与木瓜蛋白酶和碱性蛋白酶的能力相近。     

水酶法提取花生水解蛋白的研究

采用水酶法从冷榨花生饼中提取水解蛋白。以溶出的蛋白质浓度为指标,研究了提取花生蛋白的酶解工艺及条件。结果表明,先用碱性蛋白酶水解再用中性蛋白酶水解效果优于单一酶,碱性蛋白酶最适条件为：温度为55℃,pH值为9．0,加酶量为0．8％,水解时间为3h;中性蛋白酶最适条件为：温度为50℃,pH值为6．5,加酶量为0．3％,水解时间为1．5h。此条件下,蛋白提取率可达90．29％。     

微波辅助分步酶解紫苏饼粕蛋白制备鲜味肽

在微波辅助的条件下,利用碱性蛋白酶和风味蛋白酶分步对紫苏饼粕蛋白进行水解,应用正交试验确定了最佳的酶解条件。通过Sephadex G-15凝胶层析、反相高效液相色谱(reversed phase-high performance liquid chromatography,RP-HPLC)法和电子舌技术对酶解液成分与鲜味的变化进行了表征。结果表明,在微波功率400 W条件下,第1步碱性蛋白酶最佳酶解条件为酶添加量1 600 U/g、p H 10.0、微波温度60℃、微波时间35 min;第2步风味蛋白酶的最佳酶解条件为酶添加量1 600 U/g、p H 6.5、微波温度65℃、微波时间40 min,分步酶解最终水解度为44.86%。最后通过凝胶层析、RP-HPLC与电子舌表征,证明微波辅助分步酶解法快速、高效,且与单独酶解所得产物相比,其产物鲜味改善明显。     

核桃蛋白DPP-Ⅳ抑制肽的分离纯化及稳定性研究

采用5种商业蛋白酶水解核桃粕,评估水解物对二肽基肽酶4（DPP-Ⅳ）的抑制活性,优选出碱性蛋白酶作为水解用酶。研究确定了碱性蛋白酶水解核桃粕的最佳水解时间为5 h,然后通过超滤和SP Sephadex C-25阳离子交换树脂柱层析分离纯化碱性蛋白酶水解物得到核桃蛋白DPP-IV抑制肽,并对所得DPP-IV抑制肽的热稳定性、pH稳定性和模拟胃肠道消化稳定性进行了测试。结果表明,核桃蛋白DPP-IV抑制肽（025 mg/mL）DPP-Ⅳ抑制率（76.19%）比未分离纯化的水解物的提高了约3倍,其富含碱性氨基酸（含量34.36%）,尤其是精氨酸含量高达25.93%。核桃蛋白DPP-Ⅳ抑制肽在高温（121 ℃）、极端pH（pH 1.0和pH 11.0）和模拟胃肠道消化条件下,均显示出良好的稳定性,因此可用作控制血糖的功能性食品成分。     

鲢鱼骨胶原多肽的制备及其抗氧化活性研究

以鲢鱼骨为原料,经蛋白酶酶解、Sephadex G-25凝胶色谱分离纯化制备具有高抗氧化活性的胶原多肽。首先以胶原多肽得率和水解度为评价指标,从中性蛋白酶、碱性蛋白酶、风味蛋白酶、胰蛋白酶和复合蛋白酶中筛选最优酶,并采用单因素实验、响应面优化实验确定鲢鱼骨胶原多肽的最佳制备工艺;然后采用Sephadex G-25凝胶色谱进行分离纯化,得到不同分子质量多肽组分并分析其氨基酸组成;以ABTS~+·清除能力和还原能力为指标,考察鲢鱼骨胶原多肽各组分的抗氧化活性。结果表明,酶解鲢鱼骨的最优酶为复合蛋白酶,制备胶原多肽的最佳工艺:酶添加量为8 220 U/g、酶解温度为51丈、酶解时间为4.5 h,此时胶原多肽得率为49.00%;经Sephadex G-25分离得到5个组分,其分子质量为14～9 788 Da,氨基酸组成符合胶原蛋白的特征;抗氧化实验表明组分Ⅳ具有最强的ABTS~+·清除能力和还原能力,其IC_(50)/OD_(1.0)分别0.02 mg/mL和19.16 mg/mL。该研究为鱼骨资源的开发利用提供了技术参考。     

多酶法在鱼露生产工艺中的应用

应用正交试验优选了碱性蛋白酶和中性蛋白酶对青鳞鱼下脚料 (鱼头、鱼骨、内脏、鱼皮等 )的水解条件 ,在此基础上 ,同时用碱性蛋白酶和中性蛋白酶 ,再用风味酶对青鳞鱼下脚料蛋白质进行水解 ,确定了以多酶法生产鱼露的新工艺。结果表明 ,青鳞鱼下脚料中蛋白质含量达 14 8% ,经多酶水解和适当调配可制得风味浓郁的鱼露。多酶法水解蛋白质的条件为 :同时用 1 5 %碱性蛋白酶和 1 5 %中性蛋白酶在 pH 7 0、5 0℃条件下水解 12 0min后再加入 2 %风味酶继续水解 60min ;新工艺生产鱼露中必需氨基酸含量达 40 3 % ,总氮达 19mg/L ,氨基氮达 11mg/g ,占总氮的 61 0 % ,呈味氨基酸含量达 49 5 %。因此新工艺鱼露营养丰富 ,味道鲜美。     

虾加工副产品中虾油提取工艺研究

通过比较碱性蛋白酶、中性蛋白酶、风味蛋白酶、复合蛋白酶和胰蛋白酶对虾加工副产品的酶解,确定风味蛋白酶作为最佳水解酶,并确定其起始pH值为6．5,考察了酶添加量、料液比、酶解时间和温度对虾油提取率的影响,确定最优工艺：酶添加量1．0g／100g,料液比1g：8mL,酶解时间2．5h,酶解温度50℃。     

地衣芽孢杆菌A 产碱性蛋白酶的研究

研究从经过60Co 照射的东海香参水解液中分离出的地衣芽孢杆菌A 产碱性蛋白酶的产率、分离纯化与生化特性。经硫酸铵沉淀、DEAE- Sepharose 离子交换层析和Sephadex G-75 凝胶层析分离纯化后,碱性蛋白酶产率为5.8%;经SDS- PAGE 电泳测得其分子质量为35kD。酶的最适反应温度为50℃,最适pH 值为11,热稳定性较好。该蛋白酶具有一定的耐氧化能力。     

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.     

鹅血中SOD的分离工艺及其抗氧化活性研究

对鹅血中SOD的纯化及抗氧化活性进行了研究.采用丙酮沉淀法对鹅血SOD进行初步纯化、Sephadex G-100进一步精制,并利用SDS-PAGE对酶的纯度检测和分子量测定.结果表明Sephadex G-100凝胶过滤层析对鹅血SOD具有较好的纯化作用,纯化后的鹩血SOD比活力从1 144.896U/mg提高到4 226.513U/mg,SDS-PAGE凝胶电泳显示单一条带,表明已达到电泳纯,且其亚基分子量约为16KD.纯化后的SOD对邻苯三酚自氧化抗氧化活性明显.     

Purification of yeast proteinase A from fresh beer and its specificity on foam proteins

Yeast proteinase A from fresh beer was first purified with Sephadex G‐100 column chromatography and the active fractions reached to 5.3‐fold purification with 7% of yield. After purification with DEAE Sephadex A50, proteinase A activity increased to be 10.1 times of the initial with 1% of yield. When identifying the sample from chromatography by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), only one protein band with 42 kDa was observed, this indicated that the enzyme was purified. The pattern of electrophoresis of hydrolysed beer by crude proteinase A did not show lipid transfer protein (LTP) on the gel. The result of SDS‐PAGE of interaction mixture of purified proteinase A and beer also indicated that LTP was decomposed.     

Alkaline protease from Bacillus cereus VITSN04: Potential application as a dehairing agent

The objective of this work is to use protease enzyme as an ecofriendly alternative to chemicals in dehairing. An alkaline protease producing bacterium was isolated from protein-rich soil sample. The bacterium was identified as Bacillus cereus VITSN04 by 16S rRNA gene sequencing method. Growth characteristics and protease activity were studied in yeast, malt, beef, nutrient broth and soybean casein digest media and the enzyme secretion was found to correspond with growth. Maximum protease production was obtained in soybean casein digest medium at 16h with the activity of 200.1±0.68U/ml and a correlation coefficient of 0.965 between growth and enzyme production. The crude enzyme was found to have maximum activity at 30°C and pH 8.0. The protease was purified by ammonium sulphate precipitation, Sephadex G-50 and G-100 gel filtration chromatography. The purified protease was homogeneous on non-denaturing PAGE and its molecular weight was estimated to be 32kDa. The purified protease was of the serine type as it was inhibited by phenylmethylsulphonyl fluoride. The crude enzyme preparation was found to be effective in dehairing goat skins in leather processing.     

曲霉羧肽酶的分离纯化研究

采用 (NH4) 2 SO4、酒精、DE52 和SephadexG1 0 0对AspergillusspCP 1发酵液进行分离纯化 ,并检测在分离纯化过程中酶的回收率和比活力 ,最后用SDS PAGE电泳检验酶的纯度。确定 (NH4) 2 SO4饱和浓度在 60 %～ 90 %之间 ,酒精浓度为 3 5 %～ 70 %之间用于粗酶液的提纯 ;电泳得到一个清晰的条带。它们的分子质量约为 15 942u。     

Production of low antigenic cheese whey protein hydrolysates using mixed proteolytic enzymes

This study examined the effect of different proteolytic enzymes on the production of cheese whey protein (CWP) hydrolysates with low antigenicity. Four enzyme combinations (1:1) trypsin + papain W‐40 (TP), trypsin + neutrase 1.5 (TN), papain W‐40 + protease S (PP) and papain W‐40 + neutrase 1.5 (PN) were added at the rate of 1% of the CWP and it was incubated for 15, 30, 60, 90, 120 and 180 min at 50 °C. CWP hydrolysis and its non‐protein nitrogen concentrations were higher with TP and TN compared with PP and PN at all incubation times. The SDS‐PAGE revealed complete removal of α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) from hydrolysates produced by trypsin‐containing enzyme mixtures. Reverse‐phase HPLC analysis ascertained the CWP hydrolysis and SDS‐PAGE results. The lowest antigenicity in CWP hydrolysates was observed with the use of trypsin‐containing enzyme mixtures compared with other enzyme combinations. Present results suggested that TP and TN combinations were the most effective for CWP hydrolysis for the removal of β‐LG from CWP. Further research is warranted to identify the peptides in CWP hydrolysates produced with these enzyme combinations that may help enhance the utilisation of whey protein in human food. Copyright © 2007 Society of Chemical Industry     

Purification of trypsin/chymotrypsin inhibitor from jack fruit seeds

A trypsin/chymotrypsin inhibitor (JSTI) was isolated from jack fruit seeds (Artocarpus integrifolia Hook f) by ammonium sulphate fractionation and chromatography on DEAE–cellulose and Sephadex G-100. During all stages of purification, the ratio of trypsin and chymotrypsin inhibitory activities remained constant. The purified preparation was found to be homogeneous by gel filtration, polyacrylamide gel electrophoresis (PAGE) and ultra-centrifugation. From the sedimentation coefficient, S _20w value of 3·5 ± 0·15 S. the molecular weight of JSTI was calculated to be 30·00 ± 2·50 kamu. The inhibitor showed a molecular weight of 24·55 kamu on a Sephadex G-75 column when eluted with 6 M guanidine hydrochloride, Under non-denaturing conditions, JSTI exhibited anomalous behaviour on a Sephadex G-200 column. On SDS–PAGE, the inhibitor showed two major bands with molecular weights of 26·30 and 15·00 kamu and two minor bands with molecular weights of 19·50 and 12·00 kamu. The carboxyamidomethylated JSTI showed three trypsin inhibitory activity bands on PAGE, suggesting the presence of isoinhibitors.     

Fractionation and Characterization of Glycomacropeptide from Caseinate and Skim Milk Hydrolysates

Glycomacropeptide (GMP) was isolated from crystalline rennin and microbial rennet hydrolyzed caseinate and skim milk by Sephadex gel filtration, DEAE Sephadex ion exchange chromatography, Con A-Sepharose affinity chromatography and exhaustive dialysis and characterized by size exclusion and reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl sulfate gel electrophoresis (SDS PAGE). Size exclusion HPLC revealed one major GMP peak with a molecular weight of 33,000 daltons. SDS PAGE revealed a group of size-heterogeneous peptides with molecular weights of < 18,000 daltons. Reversed phase HPLC revealed one major GMP peak with several minor peptides, indicating heterogeneity with respect to hydrophobicity/polarity.     

Purification and Partial Characterization of Trypsin from the Viscera of Tropical Sierra (Scomberomorus Sierra) from the Gulf of California

Trypsin from the viscera of sierra (Scomberomorus sierra) was purified by affinity chromatography on Sepharose‐4B coupled to soybean trypsin inhibitor and characterized with respect to its purity, sensitivity to temperature, pH and inhibition. Trypsin was purified from sierra viscera with 11.9‐fold and 29.7% yield. The enzyme had a molecular weight of 25.4 kDa estimated by SDS‐PAGE and two possible trypsin isoforms were observed in activity gels. Trypsin activity was strongly inhibited by soybean trypsin inhibitor and porcine trypsin inhibitor, showing a partial inhibition by a serine protease inhibitor. The optimal activity of the enzyme was observed at pH 9 and 60C with n‐α‐benzoyl‐dl‐arginine‐p‐nitroanilide as a substrate. The enzyme maintained more than 50% of its activity in temperatures up to 50C and within the pH range of 8–10 for a period of up to 2 h.     

胰凝乳蛋白酶SplB在枯草芽孢杆菌中的重组表达及应用

通过启动子优化、宿主筛选,构建了C端带His-tag的胰凝乳蛋白酶类蛋白酶SplB表达载体,成功实现了SplB在枯草芽孢杆菌中的重组表达,对纯化的重组SplB进行酶学性质研究,并实现了重组蛋白酶SplB在重组蛋白Prx标签切割中的应用。结果表明,以枯草芽孢杆菌ATCC6051Δ10为宿主,通过启动子P₄₃介导的重组蛋白酶SplB表达活力最高(10.24 U/mL)。通过亲和层析纯化了重组表达的SplB,并进行酶学性质研究,其最适温度为40℃,最适pH值为8.5,且具有良好的温度和pH值稳定性。在离子浓度较低情况下,Co²⁺对重组SplB活力有促进作用,Mg²⁺、K⁺不影响酶活力;而Cu²⁺、Zn²⁺、Ni⁺离子抑制SplB活力;十二烷基硫酸钠极大抑制重组SplB活力。将重组SplB应用于切割重组蛋白Prx的标签,结果表明,重组SplB具有WELQ肽段标签切割作用,并且SplB浓度越高,目标条带越浓。本研究为优化SplB的重组表达及在食...