源于不同菌株的谷氨酰胺转胺酶对酪蛋白的作用研究

本实验采用SDS-PAGE电泳和凝胶成像技术对TG-K和TG-N与酪蛋白反应的最佳条件(酶用量、反应时间、反应温度、反应pH值)和作用效果进行了对比分析,从而进一步探讨了源于不同菌株的谷氨酰胺转胺酶对酪蛋白的作用和酶本身的特性研究,为该酶在乳制品加工中的应用提供了理论指导。     

酱油曲蛋白酶的分离及催化性质研究

酱油曲提取液,经硫酸铵盐析后,用Sephadex G-75柱层析分离,测定收集液中的蛋白含量并用两种方法测定蛋白酶活力。结果显示,福林法酶活力峰出现在蛋白吸收曲线第二高峰,酶解酪蛋白显示的酶活力峰值出现在蛋白含量第三高峰的左肩。表明酱油曲含有不同分子量大小的蛋白酶,分别作用于蛋白质的不同位点,分子量较大(在20 kDa到14.3 kDa之间)的蛋白酶福林法酶活力最高,蛋白酶水解酪蛋白产生的酪氨酸较多;分子量比较小(小于14.3 kDa)的蛋白酶福林法检测酶活力不高,但分解酪蛋白产生的氨基酸态氮(ANN)含量较高,表明该蛋白酶酶解酪蛋白产生酪氨酸之外的其它氨基酸或肽类较多。     

奶渣酪蛋白特性及营养价值分析

为探讨奶渣酪蛋白的理化性质和营养价值,以牦牛奶渣为原料,经过碱溶酸沉,脱除脂肪和乳糖后提取奶渣酪蛋白,利用紫外分光光度计、荧光分光光度计测定理化性质,通过酶解和质谱法鉴定奶渣酪蛋白一级序列片段和潜在活性多肽片段。结果表明：经一次碱溶酸沉提取的奶渣酪蛋白中蛋白质含量达到91.41%,在碱性条件下有较好的溶解性,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示α-酪蛋白的分子量约为30 kDa、β-酪蛋白的分子量约为26 kDa。与市面上的两种酪蛋白相比,奶渣酪蛋白的乳化能力较好,表面疏水性较高,并且具有良好的热稳定性和二级结构稳定性,但是其交联性能略差。奶渣酪蛋白含有17种氨基酸,且有6种必需氨基酸含量高于市购酪蛋白;含有129种潜在活性多肽片段,可能会在降糖、降压、抗血栓、抗衰老等功能上具有一定生理活性。     

采用改良SDS-聚丙烯酰胺电泳对酱油成曲蛋白酶的分析

用改良的SDS-聚丙烯酰胺电泳对酱油成曲中的蛋白酶进行了分析研究，结果表明：酱油成曲里有多种不同分子量的蛋白酶，分子量21kDa-200kDa，主要集中在21.6kDa-38．4kDa和N52．3kDa-200kDa。同时通过在电泳时不加酪蛋白而在培育液中加酪蛋白的方法证实了结果的可靠性。     

酶解核桃蛋白制备抗氧化肽的研究

利用木瓜蛋白酶酶解核桃分离蛋白制备小分子活性肽,并研究了不同分子量段核桃小分子肽的抗氧化性能。通过单因素实验和正交实验,确定了木瓜蛋白酶制备抗氧化肽的最佳酶解条件为:pH8.5,温度50℃,酶与底物浓度之比[E]/[S]=3:100,底物浓度[S]=3.5g/100mL,酶解时间5h。用截留分子量分别为3kDa和10kDa的超滤膜将核桃粗肽液分离成<3kDa、3～10kDa及>10kDa3个分子量段,并对不同分子量段核桃多肽的抗氧化性进行了研究,结果表明,分子量<3kDa的核桃多肽的抗氧化性大于其他两个分子量段。     

酪蛋白与明胶的酶促交联与产物的流变学性质

利用转谷氨酰胺酶对酪蛋白和明胶进行酶促混合交联。在酪蛋白与明胶比例为4︰1(质量比)时,以交联产物中羟脯氨酸质量分数为指标,采用单因素试验研究酶添加量、反应时间和温度对交联反应的影响。优化后的适宜交联条件为:底物质量浓度固定为50 g/L,酶添加量为每克蛋白质20 U,反应时间为4 h,温度为45℃。SDS-聚丙烯酰胺凝胶电泳分析表明产物中含有蛋白质聚合物。与原料酪蛋白、转谷氨酰胺酶促交联的酪蛋白相比,所得到的产物的分散液表观黏度和黏弹性均有显著的改变,表明转谷氨酰胺酶催化的酪蛋白和明胶混合可以用于改善其流变学性质。     

酪蛋白的转谷氨酰胺酶氨基葡萄糖修饰与功能性变化

在37℃、pH值为7.5和氨基葡萄糖存在下,利用转谷氨酰胺酶(EC 2.3.2.13)对酪蛋白进行交联修饰制备修饰酪蛋白;用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和高效液相色谱分析证实酪蛋白同时发生交联与糖基结合,且反应4 h时每摩酪蛋白可结合1.2摩葡萄糖.与酪蛋白相比,交联酪蛋白的溶解性质和起泡性质受损,而修饰酪蛋白产品的溶解性质得到改善,起泡性质尤其是泡沫稳定性质显著提高.在蛋白质质量浓度为1 g/L时,修饰酪蛋白的起泡能力和泡沫稳定性分别比酪蛋白提高8.6%和21%;质量浓度为100 g/L的修饰酪蛋白分散液表现出非牛顿流体特性,表观黏度显著高于交联酪蛋白或酪蛋白.     

酪蛋白的转谷氨酰胺酶酶促糖基化交联条件及产物性质

在氨基葡萄糖存在的条件下,利用转谷氨酰胺酶(EC2.3.2.13)对酪蛋白进行糖基化交联修饰。以修饰酪蛋白产物中氨基葡萄糖的导入量为指标,采用单因素试验分别考察反应体系pH值、酶添加量、反应温度和时间对修饰反应的影响。优化后的适宜修饰条件为:酪蛋白底物质量浓度为30g/L,氨基葡萄糖添加量为3mol(每kg酪蛋白中)、pH值为7.5、酶添加量为10kU(每kg酪蛋白中)、反应温度为37℃、时间4h。与酪蛋白和转谷氨酰胺酶促交联的酪蛋白相比,修饰酪蛋白产物的乳化性质和胶凝性质得到显著改善,并且体外消化性能未受到影响,表明转谷氨酰胺酶催化的糖基化交联修饰可以用于改善酪蛋白的这些功能性质。     

阿魏酸存在下酪蛋白的酶促交联与一些性质变化

在pH值为9.5,阿魏酸存在和37℃的条件下,利用辣根过氧化物酶(EC1.11.1.7)和过氧化氢对酪蛋白进行氧化交联,并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳和紫外吸收分析确认交联作用。在蛋白质质量分数为0.04%时,交联酪蛋白的乳化活性指数、乳化稳定性分别比酪蛋白提高25.2%和7.0%。利用葡萄糖酸–δ–内酯制备酪蛋白和交联酪蛋白的酸化凝胶,扫描电子显微镜观察可以看出交联酪蛋白凝胶的微观结构中空穴减少,形成了更为致密的结构。     

海参肽的提取分离及其体外抗氧化活性

以水解度为指标,试验研究了复合蛋白酶、木瓜蛋白酶、水产蛋白酶、中性蛋白酶、风味蛋白酶、胰蛋白酶和碱性蛋白酶对海参蛋白的水解能力,确定以复合蛋白酶为最佳用酶。以料液比、加酶量、酶解时间为试验因素,水解度为响应值,通过响应面法对提取工艺进行优化,结果表明该酶最佳酶解条件为:加酶量0.79%、料液比1︰5.35 (g/mL)、酶解时间3.93 h。在此条件下,海参蛋白水解度为21.38%。分别用截留分子量3 kDa和1 kDa超滤膜对海参肽进行分级分离,得到分子量大于3 kDa, 1～3 kDa和小于1 kDa 3种海参肽,将不同分子量海参肽分别在ABTS+·清除活性、还原力、DPPH·清除活性、O_2~-·清除活性4种体系下比较三者体外抗氧化能力。结果显示:分子量小于1 kDa海参肽对ABTS+·、O_2~-·清除活性最强, 1～3 kDa海参肽次之,大于3 kDa海参肽最弱;分子量大于3 kDa海参肽还原力以及对DPPH清除活性最强。     

弹性蛋白酶产生菌的分离鉴定和发酵条件研究

从污水河的土壤中采集样品,分离得到一株产胞外弹性蛋白酶活性较高且稳定的细菌。经初步鉴定属枯草芽孢杆菌(Bacillus subtilis B1),其酶活力为74.25U/ml。对其进行物理诱变,诱变后的菌株(B9)产酶活性有较大的提高。B9酶活力为120.00U/ml,较原菌株提高了1.62倍。同时研究了其产酶最适条件,产酶最适碳源为蔗糖,最适氮源为干酪素,并对碳源、氮源和生长因子进行了正交试验,研究发现氮源对产酶的影响最大。当蔗糖的用量为4%、干酪素为3%、玉米提取液为0.1%时具有较高的产酶水平。最适发酵初始pH为8.0、300ml最适摇瓶装量为40ml、最适接种量为3%。经发酵条件的优化后,B9酶活达到311.54U/ml、提高了2.59倍;较以往国内报道的酶活要高。     

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.     

The Use of Red Oncom Powder as Potential Production Media for Fibrinogenolytic Protease Derived from Bacillus Licheniformis RO3

The high cost of enzyme production is one of the barriers to successful application of enzyme in the industry. Selection of media is a critical factor for the enzyme production. Main factors for optimization of enzyme production include nutritional components and environmental conditions for growth and production of fibrinogenolytic protease. In this study, Bacillus licheniformis RO3 isolated from red oncom, an Indonesian fermented food was tested for its fibrinogenolytic protease production by using several media. Three types of media were analyzed, i.e. Luria-Bertani broth (LB), ½ LB + 1% skim milk (LBS), and ½ LB + 1% red oncom powder (LBO). Protease activity was tested by using spectrophotometric method with casein as a substrate and fibrinogenolytic activity was confirmed based on zymography assay using fibrinogen substrate. In LB media, B. licheniformis RO3 was able to produce protease with activity of 0.024 U/ml or 0.157 U/mg at 36 h fermentation. In LBS media, the highest protease activity was 0.022 U/ml or 0.152 U/mg at 48 h fermentation. The best result was shown by B. licheniformis RO3 grown in LBO media with the highest protease activity of 0.051 U/ml or 0.283 U/mg at 48 h. Zymographic profiles showed that crude enzyme from B. licheniformis RO3 consisted of six fibrinogenolytic bands with molecular weight of 20, 27, 32, 40, 70, and >140 kDa. These results indicate that red oncom powder can be used as a potential media for fibrinogenolytic protease production.     

Characterisation of potential milk coagulants from Calotropis gigantea plant parts and their hydrolytic pattern of bovine casein

The steady increase in world cheese demand keeps the search for appropriate coagulating enzymes as rennet substitutes in the scientific focus. This work reports the milk-clotting activities of aqueous crude enzyme (CE) from different parts of Calotropis gigantea using skim milk as substrate. CE from latex exhibited high caseinolytic (86.445 ± 1.055 U/ml) as well as milk-clotting activity (450 U/ml) when compared to other parts. Significant milk clotting index (MCI) was presented by crude enzyme of latex followed by stem, flower and leaf in the decreasing order (p < 0.05). CE from all parts showed an optimum activity at 37 °C, pH 5.5 and 10 mM calcium chloride concentration with excellent pH (4.5–6.5) and thermal stability (30–60 °C). Hydrolysis pattern of casein components by CE during 1 h incubation, as assessed by Tricine–SDS-PAGE, and subsequent peak analysis revealed CE from stem to be closely similar to that of rennin. However, extensive casein hydrolysis was observed when incubated with crude latex enzyme. Casein bands of CE and rennin tended to disappear as incubation progressed to 24 h. Proteolytic degradation was found to be advanced and resulted in complete breakdown of casein fractions unravelling the importance of incubation time as a possible essential requisite for controlled and appropriate casein hydrolysis. The study provides evidence of rennin-like activity associated with C. gigantea plant parts which may serve as a promising source of vegetable milk coagulant.     

Yak milk casein as a functional ingredient: preparation and identification of angiotensin-I-converting enzyme inhibitory peptides

Yak milk casein derived from Qula, a traditional Tibetan acid curd cheese, was hydrolyzed by six commercially available proteases (Trypsin, Pepsin, Alcalase, Flavourzyme, Papain and Neutrase). These hydrolysates were assayed for their inhibitory activity of Angiotensin-I-converting enzyme (ACE). The hydrolysates obtained by Neutrase from Bacillus amyloliquefaciens showed the highest ACE inhibitory activity. The IC50 value of Neutrase-hydrolysate was 0.38 mg/ml. The hydrolysate obtained by Neutrase was further separated by consecutive ultra-filtration with 10 kDa and then with 6 kDa molecular weight cut-offs into different permeated parts and fractionated by gel filtration chromatography with a Sephadex G-25 column. The active fraction was subjected to RP-HPLC, in which five peaks were purified and identified. Amino acid sequence analysis confirmed that the peptides and origins were as follows: YQKFPQY (alphas2-CN; f89-95), LPQNIPPL (beta-CN; f70-77), SKVLPVPQK (beta-CN; f168-176), LPYPYY (kappa-CN; f56-61) and FLPYPYY (kappa-CN; f55-61). Their amino acid sequences matched well with those of known bioactive peptides from bovine casein. The results indicated that yak milk casein could be a resource to generate antihypertensive peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

LiCl-ARTP复合诱变选育高产碱性蛋白酶菌株及其发酵条件优化

以地衣芽孢杆菌（Baclicus lincheniformis）E-417为出发菌株,采用氯化锂（LiCl）-常压室温等离子体（ARTP）复合诱变法对其进行诱变,通过酪蛋白平板初筛、摇瓶复筛、遗传稳定性验证筛选高产碱性蛋白酶的优良菌株,并通过单因素及响应面试验对其发酵产酶条件进行优化。结果表明,在氯化锂添加量为1.5%、ARTP照射时间为45 s的最适复合诱变条件下筛选得到1株高产碱性蛋白酶的优良菌株F-3,酶活达到12 147 U/mL,且该菌株传代8次后仍具有良好的遗传稳定性,其最适发酵培养基成分为玉米粉41 g/L、豆饼粉40 g/L、碳酸钠2.1 g/L、磷酸氢二钠2.0 g/L;最适发酵条件为发酵温度37 ℃、初始pH值7.0、接种量8%。在此优化条件下,碱性蛋白酶酶活达到16 156 U/mL,较原始菌株提高75%。     

抗氧化大豆多肽制备的研究

以还原能力、清除超氧阴离子自由基和过氧化氢能力为指标,筛选制备抗氧化大豆多肽的水解酶及水解条件,得到高效的抗氧化大豆多肽制品。结果表明:选择1000U/g菠萝蛋白酶、2000U/g中性蛋白酶、2000U/g碱性蛋白酶,三酶复合在pH6.0,酶解温度50℃,底物浓度8.0%的酶解条件下,对大豆蛋白酶解4h,多肽的抗氧化能力表现最强,还原能力达到0.898,超氧阴离子自由基的清除率达到40.93%,过氧化氢的清除率达到33.37%。经超滤分离,分子量小于10KDa而大于5KDa的多肽表现出较强的还原能力,而小于5KDa的多肽表现出较强的清除超氧阴离子自由基和过氧化氢的能力。     

Milk lipoprotein lipase distribution in the major fractions of bovine milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored less than or equal to 24 h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72 h of cold (4 degrees C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4 degrees C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)