胰蛋白酶水解全酪蛋白反应过程中的分析

将高效凝胶排阻 (HPSEC)技术与水解度 (DH)概念相结合 ,对酪蛋白 胰蛋白酶水解体系的酶解反应过程进行分析 ,得到定量表征复杂酶解反应进程和不同DH值时多样性酶解产物相对分子质量分布的二维图线 ;依据蛋白质结构信息 ,结合HPSEC实验谱图 ,对胰蛋白酶作用于酪蛋白时的酶解断裂位点进行剖析 ,初步推断反应历程 ,并得到理论酶解肽段的相对分子质量分布图及酶解物中活性多肽酪蛋白磷酸肽 (CPPs)肽谱。     

基于分子机制的蛋白质酶解反应经验修饰动力学模型

蛋白质酶解反应动力学行为的复杂性在于体系中底物与产物的多样性,以及由此决定的动力学常数的可变性.基于此,以酪蛋白(case in)—胰蛋白酶(trypsin)为模式体系,拟合求得动力学常数(Km和kcat)随水解度(DH)值变化的函数表达式,其规律为:随DH值增加,Km增大,kcat减小,kcat/Km减小,证明:酶与底物的亲和力随肽链缩短而减小,即高分子量多肽为蛋白酶的适宜底物,而酶解效率与酶解专一性随反应进行逐渐降低.进一步,根据分子水平的蛋白质酶解作用机制,关联水解实验数据,得到case in-trypsin酶解反应的经验修饰动力学方程(模型平均相对误差<5%),为定量表征复杂酶解历程以及高效制备活性多肽提供了理论基础.     

酪蛋白酶解反应复杂产物的色谱定量分析

根据酪蛋白酶解产物的高效凝胶排阻色谱(HPSEC)定性分析结果,选取不同截留相对分子质量的超滤膜对酶解产物进行截分,制得3种相对分子质量分布相对集中的区间组分;建立各组分峰面积与质量浓度之间的线性关系,由此定量计算不同反应时间的酶解产物中相应相对分子质量区间组分的质量浓度。研究表明,将膜分离方法与色谱分析相结合可实现对蛋白质、多肽等复杂体系相对精确的定量分析。     

酶催化水解法快速测定牛奶中蛋白质含量

以酪蛋白水解度为指标,采用pH-stat法优化了胰蛋白酶催化酪蛋白水解的反应条件,并以该酶为检测用酶,分析了酪蛋白浓度与酶解反应初速度的关系,并建立了一种快速检测牛奶中蛋白质含量的方法.在pH=7.5,温度55℃的条件下,用pH-stat法测得酪蛋白浓度与酶促蛋白质水解反应初速度呈良好线性关系.最后采用该酶催化水解法测...     

超高效液相色谱-串联质谱法测定牛源性酪蛋白方法中酶解条件的优化及酶解性能的评价

为提高质谱法测定蛋白质的准确性,对酶解条件的优化和选择并建立最佳条件,以期提高回收率。设计了7种酶解方法进行了比较,并引入豆浆作为阴性基质。其中直接酶解法被选为最佳方法,其操作操作过程如下:取1g豆浆粉溶于10mL的50mmol·L-1三羟甲基氨基甲烷-盐酸缓冲溶液中制成豆浆提取液,取200nmol·L-1β-酪蛋白溶液200μL,加入豆浆提取液300μL,再加10μg质谱级胰蛋白酶,混匀后于37℃恒温酶解16h,加入10%(体积分数)甲酸溶液10μL,终止反应,加水定容至1.0mL,高速离心10min,取上清液进样,按仪器工作条件测定VLPVPQK特征肽段的峰面积。此方法的平均酶解效率为106%,相对标准偏差(n=5)为5.1%。方法中选择酶的加入量w酶与β-酪蛋白量wβ-酪蛋白量之比为1∶20。在方法的优化过程中不建议进行还原和烷基化步骤。     

氢氧化钡水解HPLC法测定碘化酪蛋白中甲状腺素的含量

建立了碘化酪蛋白中甲状腺素 (T4)的氢氧化钡水解HPLC测定方法。实验发现 ,每毫升碘化酪蛋白溶液加入0.5～1.0g的八水合氢氧化钡 ,110℃水解8～12h ,控制用硫酸沉淀钡离子后的pH12～13,碘化酪蛋白释放的甲状腺素达到相对最高稳定得率。使用WatersSymmetryShieldTMRP18色谱柱 (5μm ,3.9mm×150mm) ,乙腈 (B ,0.1 %的甲酸 )和水 (A ,0.1%的甲酸 )为流动相 ,0.8mL·min -1的流速 ,B从10 %到50 %线性洗脱40min ,240nm的紫外吸收波长 ,能使碘化酪蛋白水解液中甲状腺素得到完全分离并定量测定。方法在考察的T4 质量浓度范围内 (0.5～25mg·L-1)线性良好 ,r>0.99 ,检出限可达0.2mg·g-1,实验条件下测定的商品碘化酪蛋白中T4含量为1.0 %     

DTNB标记的肌酸激酶的水解及氰解反应的研究

本文研究了DTNB标记的肌酸激酶(S,S′-二-TNB-CK)氰解反应,发现在Degani等人的氰解反应的条件下,同时有水解反应存在。当没有KCN存在时,在pH9.5的条件下,DTNB标记的CK可以迅速地被水解,并释放出TNB基团。水解反应呈双相的一级反应,在快相反应中每个酶分子大约释放一个TNB基团。当水解后的产物进一步与KCN反应时,每个酶分子大约又释放一个TNB基团,反应则呈单相的一级反应。上述结果表明在DTNB修饰酶中,两个被修饰的巯基分别处于二聚体肌酸激酶的每一个亚基的活性部位上,其中一个TNB基团在水解反应中迅速被释放出来而另一个以很慢的速度反应并很难反应完全。而水解产物进行氰解时,那个很难被水解的TNB基团此时被释放出来。这表明了肌酸激酶的两个亚基结合是不对称的,因而导致两个活性部位的巯基处于不同的化学微环境中。此外还发现在水解过程或氰解过程中伴随着TNB基团的释放,活性部位部分巯基被还原成游离巯基,同时活力也得到部分恢复并呈正相关作用。这进一步说明CK中可反应的半胱氨酸巯基是酶的必需基因,并处于酶分子的活性部位上。     

马鲛鱼抗氧化肽的纳流液相色谱法分离与筛选

在海洋天然产物中,马鲛鱼是一种重要的高活性抗氧化肽生物源,具有极高的加工附加值。由于鱼体组织的复杂性,活性抗氧化肽成分的提取和筛选对样品制备和分离技术提出了挑战。使用不同蛋白酶对鱼体组织进行酶解时,所获得的活性肽结构及功能活性会有显著的差别。为了获得高活性的抗氧化肽,该研究分别考察了风味蛋白酶、胰蛋白酶、酸性蛋白酶、中性蛋白酶、碱性蛋白酶5种蛋白酶的酶解效果。以二苯代苦味肼基自由基(DPPH·)、羟自由基(·OH)清除率和水解度(DH)为指标,筛选最优水解酶。结果表明,胰蛋白酶酶解液清除DPPH·和·OH能力最强,清除率分别达到88.93%±0.82%和53.09%±0.73%。在单因素试验的基础上,以DPPH·清除率为响应值,以加酶量、酶解温度和时间为函数,进行了三因素三水平响应面试验,获得水解度23.66%、DPPH·清除率93.78%以及·OH清除率62.59%的最优制备条件。纳流液相色谱具有低样品量、低溶剂消耗和高效等优势。为筛选出适合于马鲛鱼内脏抗氧化肽分离分析的固定相,该研究使用1∶1000分流比的纳流液相平台,分别使用反相C18柱(15 cm×100 μm, 5 μm, 30 nm)和强阳离子交换柱(15 cm×100 μm, 5 μm, 100 nm)进行分离,收集、冻干并评测了各组分的抗氧化能力。结果表明,强阳离子交换固定相更适合于马鲛鱼内脏抗氧化肽的分离纯化,并筛选出1个强活性抗氧化肽组分。该组分DPPH·清除力的半抑制浓度(IC₅₀)为0.672±0.051 mg/mL,与纯化前相比提高了13.6倍。该研究报道了纳流液相色谱在海洋天然产物源抗氧化肽分离分析中的应用,并证明了其在活性抗氧化肽成分筛选中的有效性和良好的应用前景。     

溶胶凝胶膜包埋的胰蛋白酶用于高通量蛋白质肽谱分析

采用溶胶凝胶包埋法在96孔板中固定胰蛋白酶,制备高通量的酶解反应器。考察了四乙氧基硅烷(TEOS)与甲基三甲氧基硅烷(MTMS)的不同比例对凝胶膜的成胶速度、机械强度、透明度、固定酶的活性及稳定性的影响。以牛血清白蛋白(BSA)为目标蛋白,比较了溶胶凝胶固定化酶与溶液态酶的稳定性和催化活性,用HPLC对BSA的酶解产物进行了肽谱分析,比较了二者的酶解效率。结果表明,TEOS与MTMS比例为1∶1时,凝胶膜有很好的成胶速度、机械强度、透明度;包埋态酶有很高的活性和稳定性。HPLC对肽段的分析结果表明,固定化酶的酶解效率优于溶液态酶。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测食品中的牛奶过敏原酪蛋白

基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱系统,建立了食品中牛奶过敏原酪蛋白的快速筛查和定量检测方法。样品经缓冲液提取后,采用5 kD超滤膜去除小分子杂质,得到蛋白质提取液。以数据依赖采集(data-dependent acquisition,DDA)方式获得全扫描质谱图,进行蛋白质定性确证,以平行反应监测(parallel reaction monitoring,PRM)技术对目标特征肽段进行定量分析。针对特征肽段,设计并合成了内标肽和内标物质,以降低基质效应和抵消处理过程中的损失。该方法应用于食品中的α-酪蛋白、β-酪蛋白和κ-酪蛋白的快速筛查和定量检测。结果表明,该方法在5~250μg/L范围内线性关系良好,定量限为0.2~5.5μg/kg,平均回收率在68.8%~104.4%之间,RSD6%。该方法可用于果汁饮料、果酱、面包、早餐谷物中牛奶过敏原酪蛋白的快速筛查和定量分析。     

Quantitative analysis of complex casein hydrolysates based on chromatography and membrane

The enzymatic hydrolysates of casein are so complex that there is no effective method to do quantitative analysis. The common techniques, such as high performance chromatography and SDS-PAGE, can only carry out qualitative analysis. On the basis of membrane separation and high performance size exclusion chromatography (HPSEC), standard peptides with different molecular mass range were prepared, and the linear relationships between mass concentration of the standard peptides and the ultraviolet absorption of corresponding peak areas were established. Consequently, mass concentration of the different hydrolysates at different reaction times could be accurately calculated. The combination of chromatography and membrane separation is of great importance to the quantitative analysis of the complex hydrolysates, which can also be applied to the other macromolecular systems, such as carbohydrates. __________ Translated from Chinese Journal of Chromatography, 2005, 23(2) (in Chinese)     

Antioxidant Activity Evaluation of Oviductus Ranae Protein Hydrolyzed by Different Proteases

As nutrition and a health tonic for both medicine and food, the protein content of Oviductus Ranae is more than 40%, making it an ideal source to produce antioxidant peptides. This work evaluated the effects of six different proteases (pepsin, trypsin, papain, flavourzyme, neutral protease and alcalase) on the antioxidant activity of Oviductus Ranae protein, and analyzed the relationship between the hydrolysis time, the degree of hydrolysis (DH) and the antioxidant activity of the enzymatic hydrolysates. The results showed that the antioxidant activity of Oviductus Ranae protein was significantly improved and the optimal hydrolysis time was maintained between 3–4 h under the action of different proteases. Among them, the protein hydrolysate which was hydrolyzed by pepsin for 180 min had the strongest comprehensive antioxidant activity and was most suitable for the production of antioxidant peptides. At this time, the DH, the DPPH radical scavenging activity, the absorbance value of reducing power determination and the hydroxyl radical scavenging activity corresponding to the enzymatic hydrolysate were 13.32 ± 0.24%, 70.63 ± 1.53%, 0.376 ± 0.009 and 31.96 ± 0.78%, respectively. Correlation analysis showed that there was a significant positive correlation between the hydrolysis time, the DH and the antioxidant activity of the enzymatic hydrolysates, further indicating that the hydrolysates of Oviductus Ranae protein had great antioxidant potential. The traditional anti-aging efficacy of Oviductus Ranae is closely related to the scavenging of reactive oxygen species, and its hydrolysates have better antioxidant capacity, which also provides support for further development of its traditional anti-aging efficacy.     

Molecular mass distribution of sodium alginate by high-performance size-exclusion chromatography

A sensitive high-performance size-exclusion chromatography (HPSEC) method with simple UV detection was developed for the molecular mass analysis of sodium alginate. It was used to evaluate alginates of varying molecular mass and the results were compared with the viscosity measurements. This HPSEC method was sensitive to serve as the stability indicating method for alginate after storage under different conditions. The information of relative molecular mass distribution of alginate was provided with reference to pullulan molecular mass standards. The comparison of the HPSEC chromatograms of alginate, pullulan and dextran revealed the effect of chemical composition of a polysaccharide and its effect on apparent molecular mass distribution.     

A Two-Step,One-Pot Enzymatic Method for Preparation of Duck Egg White Protein Hydrolysates with High Antioxidant Activity

Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8–10 % and soluble peptide yield (SEP) of 60.5–70.2 % in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2 % and 90.4 %, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21 % gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis.     

The Influence of the Extent of Enzymatic Hydrolysis on Antioxidative Properties and ACE-Inhibitory Activities of Protein Hydrolysates from Goby (Zosterisessor ophiocephalus) Muscle

Antioxidant properties and angiotensin-converting enzyme (ACE) inhibitory activities of protein hydrolysates from goby (Zosterisessor ophiocephalus) muscle, with different degrees of hydrolysis (DH) from 5 to 25 %, prepared by treatment with crude proteases extract from smooth hound intestines, were investigated. Goby protein hydrolysates (GPHs) are rich in Gly and Thr, which accounted for 14.1–15 % and 11.6–13.2 % of the total amino acids, respectively. The antioxidant activities of GPHs were investigated by using several in vitro assay systems. All GPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. For the ACE-inhibitory activity, as the DH increased, the activity of GPHs increased. The obtained results revealed that antioxidant and ACE-inhibitory activities of GPHs were influenced by the degree of hydrolysis. A medium degree of enzymatic hydrolysis was appropriate to obtain GPHs with good antioxidant activity, while small peptides were essential to obtain high ACE inhibitory activity.     

Fractionation of casein hydrolysates using polysulfone ultrafiltration hollow fiber membranes

The objective of this study was to characterize the fractionation profile of casein hydrolysates obtained with polysulfone hollow fiber ultrafiltration membranes. The two-step ultrafiltration process developed by Turgeon and Gauthier [J. Food Sci., 55 (1990) 106] was used: a caseinate solution was submitted to proteolysis with chymotrypsin or trypsin, and the reaction mixture (RM) was subsequently ultrafiltered using a 30 kDa (MWCO) hollow-fiber polysulfone membrane. The total hydrolysate permeating from this first step was further fractionated using a 1 kDa (MWCO) membrane, producing the mixture of polypeptides (retentate) and the amino acid fraction (permeate). The effect of enzyme specificity and of membrane retentivitiy on the total composition (total nitrogen, fat, lactose, minerals) and amino acid profile of the fractions was studied. The overall composition of the fractions was not significantly affected by the nature of the enzyme but the degree of hydrolysis and the molecular weight distribution profile analyses showed a marked effect of the enzyme specificity, with trypsin giving a larger proportion of small peptides (< 200 Da) in the mixture of polypeptides. Amino acid profile analyses provided useful information on the phenomena governing the fractionation of amino acids with a polysulfone membrane: (1) the target amino acids of the enzyme are concentrated in the permeate as a result of their presence in all peptides produced by hydrolysis, (2) polar amino acids are retained by the membrane, (3) non-polar amino acids are not selectively rejected by the membrane. Our results suggest that the charge/hydrophobicity balance of the peptides produced is the predominant factor determining the fractionation of casein hydrolysates.     

Optimization of a microwave-coupled enzymatic digestion process to prepare peanut peptides

The best enzyme to prepare peanut peptides, papain, coupled with microwave irradiation was selected from five common proteases according to the results of the yield of peanut peptides [nitrogen solution index (NSI) in trichloroacetic acid (TCA), TCA-NSI] and the degree of hydrolysis (DH). The main factors that influenced the microwave-coupled enzymatic digestion method were optimized by response surface analysis. The optimal conditions obtained were as follows: microwave extraction time, 9.5 min; power, 600 W; substrate concentration, 4%; enzymatic reaction temperature, 50 °C; enzyme quantity, 6,500 U/g; pH, 7.1 (phosphate buffer, 0.05 mol/L). Under these conditions, TCA-NSI was 62.00% and DH was 25.89%, which is higher than that obtained with either protease hydrolysis or microwave hydrolysis alone.     

Characterisation of legumes by enzymatic hydrolysis,microdialysis sampling,and micro-high-performance anion-exchange chromatography with electrospray ionisation mass spectrometry

An assay based on enzymatic hydrolysisand microdialysis sampling, micro-high-performance anion-exchange chromatography (micro-HPAEC) with electrospray ionisation mass spectrometry (ESI-MS) for the characterisation of legumes is presented. Characterisation of two bean varieties; Phaseolus mungo and P. acutifilous was based upon enzymatic hydrolysis using an endo-beta-mannanase from Aspergillus niger with subsequent analysis of the hydrolysates with HPAEC-MS. The hydrolysates were detected in the positive ionisation mode after desalting the chromatographic effluent, employing a cation-exchange membrane desalting device with water as the regenerating liquid. Mass chromatograms, acquiredafter hydrolysis of both bean samples for 12 h, showed two different profiles of hydrolysates. The P. mungo bean hydrolysate showed the presence of saccharides with a degree of polymerisation (DP) in the range of 2-6, whereas that of P. acutifilous showed only DPs of 2-5. Both bean samples had one type of DP 2, but showed different types of DPs 3, 4 and 5. Only the P. mungo sample showed the presence of DP 6. The most abundant fraction for P. mungo was DP 4, whereas that for P. acutifilous was DP 5. Tandem MS of the hydrolysates showed that the DP 2 hydrolysates observed for the samples were of the same type, having a 1,6 linkage. Also tandem MS data for DPs 3, 4, and 5 showed that similar hydrolysates were present within the same sample as well as among the two samples. The data also showed the existence of 1,6 linkages for DP 3, 4, and 5 hydrolysates. The single enzymatic hydrolysis in combination with microdialysis and HPAEC with ESI-MS proved to be sufficient and reproducible for profiling and showing the difference between the two bean samples.