木瓜蛋白酶水解酪蛋白制备降血压肽的工艺优化

目的:以酪蛋白为原料,酶解法制备降血压肽,优化工艺。方法:采用5种不同蛋白酶水解酪蛋白,根据水解产物的血管紧张素转化酶(ACE)体外抑制活性选择蛋白酶。以酶用量、酶解时间、底物浓度3因素作为研究对象,以可溶性蛋白产量、ACE体外抑制活性两参数为评价指标,通过Box-Behnken响应面分析法优化水解工艺。结果:木瓜蛋白酶水解酪蛋白的产物活性最高,因此作为工艺优化用酶。响应面分析法得到的最优工艺条件为:酶用量0.032 g,酶解时间4.53 h,底物浓度0.70%,可溶性蛋白产量1283.71 mg、ACE抑制率为87.23%。结论:Box-Behnken设计可有效优化木瓜蛋白酶水解酪蛋白制备降血压肽工艺,所得工艺稳定可靠。     

木瓜蛋白酶水解羊乳酪蛋白的工艺研究

目的:研究木瓜蛋白酶酶解羊乳酪蛋白的最优工艺条件,以获得具有抗菌活性的酶解物.方法:以山羊乳为原料制备酪蛋白,以木瓜蛋白酶为水解酶对其进行水解,通过单因素试验和正交试验,以酶解液中氨基态氮含量为指标,优选木瓜蛋白酶酶解山羊乳酪蛋白的最佳工艺条件,并用该酶解物做抑菌活性试验.结果:木瓜蛋白酶酶解山羊乳酪蛋白的最佳酶解工艺:酶与底物比4 000 U/g,底物质量浓度85 g/L,初始pH 6.0,在温度60 ℃下酶解150 min.抗菌试验结果表明,酶解液氨基氮质量浓度为0.2889 g/100 mL时,对Ecoli.O157有一定的抑菌活性.结论:用木瓜蛋白酶酶解山羊乳酪蛋白,可得到具有一定抑菌活性的酶解物.这是获得抗菌肽的良好来源.     

高活性酪蛋白抗氧化肽的制备方法研究

采用木瓜蛋白酶对酪蛋白进行水解,得到抗氧化活性较好的酪蛋白水解物,并且水解物在木瓜蛋白酶作用下进行类蛋白反应制备出高活性酪蛋白抗氧化肽。第一步制备酪蛋白水解时酶添加量为500 U/g酪蛋白、温度45℃、底物浓度5%、反应时间2 h。第二步类蛋白反应的最优条件为：酶添加量为500 U/g水解物、温度30℃,底物浓度50%、作用时间5.5 h。毛细管电泳结果确认,类蛋白反应修饰后抗氧化肽的组成情况发生变化。抗氧化活性分析结果表明,类蛋白反应修饰后的酪蛋白抗氧化肽对两种自由基的清除能力显著提高。     

酶解条件对鹰嘴豆多肽抗氧化活性的影响

以鹰嘴豆蛋白粉为原料制备鹰嘴豆蛋白抗氧化活性肽,采用中性蛋白酶、木瓜蛋白酶和碱性蛋白酶分别对鹰嘴豆蛋白进行酶解,测定了不同酶作用下的鹰嘴豆多肽抗氧化活性,确定木瓜蛋白酶是酶解鹰嘴豆蛋白的最适蛋白酶。研究了温度、p H、底物浓度、酶添加量等酶解条件对产物抗氧化活性的影响。结果表明,不同的酶解条件对鹰嘴豆多肽的抗氧化活性具有显著影响,最优酶解条件为:温度60℃,p H 6.0,底物浓度3.0%,酶的添加量3.0%,在此条件下酶解180 min,制得的鹰嘴豆多肽具有较高的抗氧化活性,其多肽含量高达71.31%。     

酪蛋白糖巨肽酶解物的二肽基肽酶-4抑制作用

为探讨酪蛋白糖巨肽(GMP)酶解物的二肽基肽酶-4(DPP-4)抑制作用,选择木瓜蛋白酶、碱性蛋白酶、胰蛋白酶、胃蛋白酶分别酶解GMP获得糖巨肽酶解物,选择甘氨酰脯氨酸对硝基苯胺为底物的发色底物法检测DPP-4活性。结果表明,GMP酶解物的DPP-4抑制效果优于GMP本身,并且随酶解时间的延长酶解物DPP-4抑制作用逐渐增加,其中GMP木瓜蛋白酶解物对DPP-4抑制效果最好,GMP木瓜蛋白酶酶解物作为DPP-4抑制剂具有潜在应用价值。     

木瓜蛋白酶制备广昌白莲降血压活性肽的研究

为探讨木瓜蛋白酶对广昌白莲蛋白的水解特性及所获多肽的降血压活性,以广昌白莲蛋白为原料,以水解度(hydrolysisdegree,DH)为指标,选用木瓜蛋白酶水解白莲蛋白获得多肽,通过测定多肽对血管紧张素转换酶(angiotensin converting enzyme,ACE)的抑制活性,得到广昌白莲ACE抑制活性肽.通过正交实验Ln(34)因素水平实验法确定木瓜蛋白酶水解白莲的最优条件为pH6.5,温度55℃,底物浓度5％,酶浓度8％.由极差值R可知各因素的主次关系是温度＞pH＞底物浓度＞酶浓度.对不同水解度下的多肽进行了降血压活性的研究,结果表明,ACE抑制率与水解度呈正相关.对多肽进行大孔吸附树脂静态乙醇分级洗脱后测定降血压活性,结果表明,经25％乙醇洗脱后,多肽降血压活性最高,为55.78％,其IC50为0.045mg/mL.     

木瓜蛋白酶酶解核桃粕蛋白产物抗氧化活性研究

目的:研究木瓜蛋白酶酶解核桃粕蛋白产物的抗氧化特性.方法:测定不同酶解时间下的酶解液的抗氧化性;用葡聚糖凝胶分离活性肽段并测定其抗氧化性.结果:在酶用量6 000 U/g 底物、底物质量分数3%、pH7.5、温度55℃的酶解条件下酶解210 min,酶解液的抗氧化活性较高,0H·清除率55.43%,O2-·清除率64.23%,DPPH·清除率79.32%,总抗氧化能力98.74%单位/mL;通过葡聚糖凝胶分离并与已知标准品对照,木瓜蛋白酶酶解核桃粕蛋白产物抗氧化活性多肽的分子质量主要集中在1321～607 Da,该分子质量片段活性多肽对0H·的清除率为42.26%,对02-·的清除率为57.33%,对DPPH·清除率为74.43%,总抗氧化能力88.43单位/mL.结论:木瓜蛋白酶酶解核桃粕蛋白210 min时酶解液的抗氧化活性较高,抗氧化活性多肽分子质量主要集中在1 321～607 Da之间.     

蚕蛹蛋白酶解工艺及其水解产物抗氧化性

以蛋白水解度为指标,利用中性蛋白酶和木瓜蛋白酶酶解蚕蛹蛋白。通过单因素试验和正交试验对影响蚕蛹蛋白水解度的因素进行优化试验,分析了两种蛋白酶对蚕蛹蛋白酶解效果的影响,并测定水解产物抗氧化活性。结果表明,中性蛋白酶酶解蚕蛹蛋白时,最优工艺条件为底物浓度为1.2%、酶解时间3.5 h、酶解pH 10.0、酶与底物比5.0%,此时,蚕蛹蛋白水解度最高为27.38%;木瓜蛋白酶酶解蚕蛹蛋白时,最优工艺条件为底物浓度为1.5%、酶解时间5.0 h、酶解pH 10.0、酶与底物比9.0%,此时,蚕蛹蛋白水解度最高为18.26%。最佳工艺条件下,中性蛋白酶和木瓜蛋白酶酶解产物DPPH清除率分别为83.84%和81.24%,超氧阴离子自由基清除率分别为6.36%和9.72%,羟基自由基清除率分别为43.30%和62.24%。综上所述,木瓜蛋白酶对酶解产物的抗氧化效果较好。     

条斑紫菜蛋白酶解物降血压活性

研究了以条斑紫菜为原料,采用木瓜蛋白酶制备紫菜蛋白活性寡肽,以ACE抑制率为评价指标,研究不同水解条件下酶解液的降血压活性,优化酶解工艺条件并测定酶解物的分子量。优化后的木瓜蛋白酶酶解工艺条件为pH7.5,温度50℃,底物浓度30 mg/mL,酶添加量600 U/g,在此条件下,水解4 h的酶解产物抑制活性达30%以上,ACE抑制肽的半抑制浓度IC50值为4.48 mg/mL。将制备的酶解液进行层析分析,测得具有降血压活性的紫菜蛋白酶解产物是相对分子质量小于2 000的活性肽。     

醇类有机溶剂对木瓜蛋白酶催化活性的影响机理

为了探索木瓜蛋白酶在有机溶剂中的催化机理和活力变化,为其工业应用打下基础,研究了在4种不同有机溶剂极性系数的醇类单相共溶体系中,木瓜蛋白酶水解酪蛋白的催化活性及木瓜蛋白酶构象的变化。结果表明:木瓜蛋白酶在体积分数15%丙二醇和体积分数8%正丙醇的溶液中水解酪蛋白的活性分别上升20.1%和17.8%,在乙二醇和丙三醇体系中则酶活力显著下降。动力学测定表明,木瓜蛋白酶在4种有机介质中水解酪蛋白的米氏常数Km与Vmax均明显下降。差示光谱和荧光发射光谱表明,体积分数40%丙三醇可使木瓜蛋白酶含有Trp残基区域的空间结构趋向更紧密,而在体积分数15%丙二醇和体积分数10%乙二醇体系中,无规则卷曲或者β-折叠增加的区域很少包含Trp残基。     

响应面法优化预调理火锅牛肉酶法嫩化工艺

针对预调理火锅牛肉的嫩化工艺,采用木瓜蛋白酶和中性蛋白酶进行复配,研究2种酶的复配比例、复合酶添加量及酶处理时间对牛肉持水力和硬度的影响.在单因素试验基础上,采用响应面优化设计对工艺条件进行优化,确定复配酶法嫩化最佳工艺条件为:木瓜蛋白酶和中性蛋白酶复配比例1:1.19、复合酶添加量0.085％、酶处理时间3.1?h,...     

THE KINETIC PROPERTIES OF LACTASE,PAPAIN AND LIPASE IMMOBILIZED ON A SINGLE SUPPORT1

Lactase, papain and lipase enzymes were immobilized concomitantly on derivatized Sepharose 4-B. Equal molar concentrations of each enzyme were allowed to react with activated Sepharose and a preferential binding of lipase and papain over lactase was observed. This preferential binding was explained by differences in diffusion rates of enzymes resulting in greater availability of binding sites within the beads to certain enzyme species. The effects of pH of assay, temperature of assay and substrate concentrations on each individual enzyme were determined. Soluble forms of each enzyme were used for comparison. There were some differences in pH and temperature optima for the immobilized enzymes. However, the affinity of the enzymes for their substrates was substantially the same.     

Improving enzyme immobilization in mesocellular siliceous foams by microwave irradiation

Microwave irradiation was used to immobilize papain and penicillin acylase in mesocellular siliceous foams (MCFs) at low temperature. The maximum loading of papain reached 984.1 mg/g, 1.26 times that obtained using the conventional, non-microwave-assisted method. The half-life (t(0.5)) of papain immobilized in MCFs by microwave irradiation at 80 degrees C was 17 h, 5.21 times that of papain immobilized by conventional means. The activities of papain and penicillin acylase immobilized with the microwave-assisted method were 779.6 U/mg and 141.8 U/mg respectively, 1.86 and 1.39 times of those obtained without microwave immobilization. Using microwave irradiation it only took 140 s for penicillin acylase, an enzyme of large dimensions, to be immobilized in MCFs. In contrast, it took 15 h to do the same using the conventional method. The results showed that microwave irradiation improved the adsorption and immobilization of enzymes in mesocellular siliceous foams.     

几种蛋白酶对鮟鱇鱼骨中生物活性钙富集的比较分析

实验采用选取中性蛋白酶、木瓜蛋白酶和胰蛋白酶作用于鮟鱇鱼骨，研究了富集鮟鱇鱼骨中生物活性钙的最佳工艺条件。研究表明中性蛋白酶、木瓜蛋白酶和胰蛋白酶具有较好的富钙效果，而胃蛋白酶的富钙的效果较差。三种酶的最好作用时间为6h～8h；木瓜蛋白酶的作用的pH为7．0，温度为45℃，浓度为1500IU／s原料；中性蛋白酶的较好作用pH为7．0，温度为50℃，浓度为2500IU／g原料；胰蛋白酶的较好作用pH为8．0，温度为45℃，浓度为2000IU／g原料。     

蚕蛹蛋白酶解制备抗氧化肽的初步研究

将蚕蛹通过粉碎和丙酮脱脂后,得到蚕蛹蛋白粉.用碱性蛋白酶、中性蛋白酶、胃蛋白酶、木瓜蛋白酶、胰蛋白酶酶解蚕蛹蛋白粉,以DPPH清除能力为指标对酶解过程进行分析,结果表明胃蛋白酶对蚕蛹蛋白有较好的酶解效果.然后通过正交优化胃蛋白酶的酶解条件,并研究水解产物多肽的体外抗氧化活性.     

双酶水解提取牛蒡菊糖的研究

采用双酶水解法提取牛蒡菊糖。首先从8 种酶中选取3 种对牛蒡菊糖提取率最高的酶,分别为木瓜蛋白酶、植物蛋白酶和酸性蛋白酶,对牛蒡菊糖的提取率分别为8.83%、8.67% 和8.21%。然后对每一种酶采用单因素试验方法研究pH 值、固液比、加酶量、温度以及时间对牛蒡菊糖提取率的影响;再通过3 种酶之间的相互组合试验,选出一组最佳组合为：木瓜蛋白酶+ 植物蛋白酶,其提取率为11.43%。最后采用单因素和正交试验设计方法,研究加酶量、固液比、温度、时间以及pH 值对牛蒡菊糖提取率的影响,得到双酶水解提取牛蒡菊糖的最佳条件组合为：在木瓜蛋白酶加酶量10%、温度50℃、pH7、时间4h、固液比1:15(m/V)的条件下进行酶解,4h 后加入植物蛋白酶,加酶量20%、温度45℃、pH8,时间4h、固液比1:15(m/V),提取液经乙醇沉淀、真空浓缩,得到粗菊糖,菊糖提取率为13.41%,产品中菊糖含量为67.86%,蛋白质含量为1.32%。     

Application of enzyme in aqueous extraction of sesame oil

The oil recovery of the traditional process of extracting sesame oil by aqueous method could be improved by adding enzyme. Sesame oil was extracted under aqueous extraction conditions using three kinds of enzymes, which were papain, trypsin and cellulase. The optimal conditions were pH 7.0, 50 °C and extraction time 3 h with constant shaking at 80 rpm. The enzyme dosage and the enzyme proportion were researched by orthogonal experiment. The result indicated that the enzymes significantly improved the oil recovery. When the enzyme hydrolysis was performed with complex enzyme of papain 6,000 U, trypsin 400 U and cellulase 250 U/g oilseed, higher oil recovery (87.58 %) was achieved than the traditional aqueous extraction processing, and the oil was extremely fragrant. Moreover, the clear oil could fall directly from the centrifuge tube which greatly reduced the difficulty of separating oil.     

响应面法优化紫贻贝蛋白酶解工艺条件

分别采用中性蛋白酶、碱性蛋白酶、酸性蛋白酶、复合蛋白酶、木瓜蛋白酶、胰蛋白酶和风味蛋白酶7种蛋白酶对紫贻贝蛋白的酶解工艺条件进行研究。根据水解度和感官评定的结果,确定中性蛋白酶、复合蛋白酶、木瓜蛋白酶和风味蛋白酶可以作为紫贻贝蛋白酶解的外加蛋白酶。将上述4种蛋白酶进行两两复配,通过试验确定复合蛋白酶与中性蛋白酶按1:1进行复配,可作为紫贻贝蛋白酶解的最适复配酶。采用响应面优化分析得出复配蛋白酶最佳酶解条件为酶解时间2h、pH7、酶解温度50℃、酶添加量0.4%,在此条件进行实验,测得水解度为70.25%,游离氨基酸总量增加了388.46%。     

Antioxidant,lipase and ACE-inhibitory properties of camel lactoferrin and its enzymatic hydrolysates

The antioxidant and the lipase and the angiotensin-converting enzyme (ACE) inhibitory properties of camel lactoferrin and its hydrolysates elaborated with four proteolytic enzymes (trypsin, α-chymotrypsin, pancreatin and papain) were assessed. Lactoferrin was purified from camel colostrum using cation exchange chromatography. Camel lactoferrin hydrolysates showed different degrees of hydrolysis, reverse phase-HPLC profiles and molecular weight distributions, reflecting heterogeneity in terms of polarity and molecular weight of the generated peptides. Camel lactoferrin hydrolysates exhibited higher antioxidant, lipase and ACE inhibitory activities than native lactoferrin. Pancreatin-generated hydrolysates showed the highest lipase inhibitory activity (48.1%), while papain-generated hydrolysates presented the greatest ACE inhibitory activity (89.14%).     

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow‐up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two‐dimensional zymography on gelatin‐containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI‐ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two‐dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI‐ToF MS analysis of the zymographic spots. Copyright © 2010 Society of Chemical Industry