乙醇溶液对木瓜蛋白酶催化活性的影响

研究了乙醇溶液对木瓜蛋白酶水解酪蛋白的催化活性及构象的影响。结果表明,木瓜蛋白酶在一定浓度乙醇溶液中水解酪蛋白的活性有显著上升。动力学测定表明木瓜蛋白酶在乙醇溶液中米氏常数（Km）下降。差示光谱显示,在乙醇溶液中木瓜蛋白酶的二级结构发生了变化。荧光发射光谱表明,木瓜蛋白酶在乙醇溶液中发射峰位几乎没移动,但发射强度明显增高。     

甲醇溶液对木瓜蛋白酶催化活性的影响

试验结果表明,木瓜蛋白酶在一定浓度甲醇溶液中水解酪蛋白的活性显著上升;动力学测定表明,该酶在甲醇溶液中Km下降;在甲醇溶液中木瓜蛋白酶催化酪蛋白水解反应的最适pH为8.5,最适温度为70℃;紫外差示光谱研究表明,在甲醇溶液作用下木瓜蛋白酶的二级结构发生了变化;荧光发射光谱研究表明,木瓜蛋白酶在甲醇溶液中发射峰位向低波长移动,荧光峰值明显增高。     

木瓜凝乳蛋白酶、木瓜蛋白酶和木瓜蛋白酶Ⅲ的蛋白水解活性

番木瓜(Carica papaya)浆汁中含有三种蛋白酶——木瓜蛋白酶,木瓜疑乳蛋白酶和木瓜蛋白酶Ⅲ(以前称为肽酶)。本研究旨在确定木瓜凝乳蛋白酶和木瓜蛋白酶Ⅲ的蛋白水解活性在量和专一性方面是否与木瓜蛋白酶相同。在测试中用偶氮酪蛋白、血清清蛋白和软骨蛋白多糖作底物,精确比较标准酶制剂的水解蛋白活性;以清蛋白和磷酸化酶α作底物,比较它们的相对专一性;把蛋白质中的肽键与合成底物的水解动力学参数加以比较。     

超高压酶解酪蛋白及其产物在动物细胞培养中的应用

以牦牛酪蛋白为底物,游离氨基酸含量为指标,在超高压条件下分别采用胰蛋白酶、胃蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和胰酶六种不同的酶水解酪蛋白并筛选出最佳用酶,并在单因素实验的基础上采用正交实验确定最佳用酶水解酪蛋白的最佳工艺条件。最后探讨水解产物在细胞培养中的应用。结果表明,胰酶水解效果最好,用其在底物浓度15%,E/S=1∶5、水解压力150 MPa,温度为45℃,pH 7.5条件下水解4h后水解产物中游离氨基酸含量高达52.18%。酶解产物对BHK-21细胞没有细胞毒性,而具有促生长作用。这为酪蛋白的进一步开发利用和蛋白质水解产物在细胞培养中的应用奠定基础。     

纤维素固相化木瓜蛋白酶

 本文用叠氮法制备了纤维素固相化木瓜蛋白酶(简称CMCP)。与相应酶液水解酪蛋白的反应相比,它表现出较低的酶活性,较高的最适pH值和较高的稳定性。CMCP的比活回收率约为24％,最适pH值向碱性范围移动约为0.5个单位。CMCP经60℃热处理,持续3h活性无明显下降,在4℃下保存127天,活性只下降了40％左右。对这些参数,本文都根据CMCP的结构特点进行了分析。 CMCP柱还表现出明显的对啤酒的防浊能力。过柱的啤酒,氨基酸的含量大大增加。     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

从半夏中提取的胰蛋白酶抑制剂及其特征

应用硫酸铵分级、无离子水沉淀、2.5%TCA 处理、DEAE-Sephadex A-50离子交换层析以及胰蛋白酶-琼脂糖凝胶亲和层析,从植物半夏新鲜块茎中分离纯化到一种胰蛋白酶抑制剂。半夏抑制剂只抑制胰蛋白酶对酰胺、酯、血红蛋白和酪蛋白的水解,不能抑制胰凝乳蛋白酶、舒缓激肽释放酶、枯草杆菌蛋白酶和木瓜蛋白酶对各自底物的水解。抑制剂对猪胰蛋白酶水解酰胺、酯、血红蛋白和酪蛋白的重量抑制比值分别为1∶0.71、1∶0.88、1∶0.71和1∶0.71。从分子大小的范围看,半夏胰蛋白酶抑制剂应属大分子抑制剂。     

几种蛋白酶对文蛤肉的酶解工艺条件研究

以文蛤为原料,水解度为指标,从胰蛋白酶,木瓜蛋白酶,胃蛋白酶和风味蛋白酶中选出水解效果较好的胰蛋白酶和木瓜蛋白酶.并通过实验确定了胰蛋白酶,木瓜蛋白酶单酶水解及两者组合复合酶解文蛤肉的最佳工艺.结果表明:复合水解效果最佳.最佳工艺:先添加胰酶6000 u/g(原料),水解温度50℃,固液比1:3(V/W),pH 8.0,在此条件下水解6 h;然后改变条件,温度55℃,pH 5.5,底物浓度1:3(V/W),添加木瓜蛋白酶2000 u/g(原料),在此条件下水解2 h.通过实验验证,胰蛋白酶和木瓜蛋白酶组合在该条件下对文蛤肉蛋白具有较好的水解效果,其水解度为28.15%.     

体外法探究不同氨基酸形式酪蛋白水解物对猪小肠微生物的影响

【目的】通过体外法探究猪小肠不同肠段肠腔微生物和肠壁微生物对两种不同氨基酸形式的酪蛋白水解物的发酵特性。【方法】以生长猪的十二指肠、空肠、回肠肠腔食糜或者肠壁微生物为接种物,分别以酸水解酪蛋白(以游离氨基酸为主)和酶水解酪蛋白(以小肽为主)为底物,37°C厌氧培养发酵,于0、3、6、12 h采样,测定微生物蛋白(MCP)以及用real time-PCR进行菌群分析。【结果】(1)肠腔微生物发酵不同酪蛋白水解物:十二指肠和回肠酶水解酪蛋白组MCP的含量显著高于酸水解酪蛋白组(P0.05)。十二指肠酶水解酪蛋白组总菌、Firmicutes数量显著高于酸水解酪蛋白组(P0.05)。回肠发酵6 h后,酶水解酪蛋白组Escherichia coli和Firmicutes的数量显著高于酸水解酪蛋白组(P0.05);发酵12 h后,酶水解酪蛋白组总菌、Lactobacillus、E.coli的数量均显著高于酸水解酪蛋白组(P0.05)。(2)肠壁微生物发酵不同酪蛋白水解物:发酵12 h后,十二指肠和回肠酶水解酪蛋白组MCP含量显著高于酸水解酪蛋白组(P0.05)。十二指肠酶水解酪蛋白组Lactobacillus、Firmicutes数量分别极显著、显著高于酸水解酪蛋白组;回肠Firmicutes数量在酶水解酪蛋白组显著高于酸水解酪蛋白组(P0.05)。【结论】十二指肠和回肠的肠腔和肠壁微生物都能够利用小肽,且在一定程度上对小肽的利用更具优势。     

双酶法制备螺旋藻多肽的工艺研究

选用碱性蛋白酶和木瓜蛋白酶结合的双酶法对螺旋藻蛋白进行水解。其中,对木瓜蛋白酶水解螺旋藻蛋白的工艺进行优化。以水解度为指标,研究了酶解时间、酶与底物比、pH和酶解温度4种因素对酶解反应的影响。在此基础上设计了3因素(加酶量、酶解温度和pH)3水平的响应面试验。结果表明碱性蛋白酶水解螺旋藻蛋白的最佳酶解条件为:加酶量4300 U/g,pH 7.0,酶解温度55℃,酶解时间160 min;木瓜蛋白酶的最佳酶解条件为:酶底比为4.5%,酶解温度60℃,pH 6.5,酶解时间210 min。利用碱性蛋白酶和木瓜蛋白酶结合的双酶法制得的多肽水解度可达32.90%,与单酶法相比,水解度明显提高。     

Effect of polyethylene glycols on the function and structure of thiol proteases

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.     

Effect of ethylene glycol and polyethylene glycol on the acid-unfolded state of trypsinogen

Effect of increasing concentrations of two of the polyols, ethylene glycol (EG) and polyethylene glycol (PEG), was studied by near and far circular dichroism (CD), fluorescence emission spectroscopy, and binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show the transition of acid-unfolded trypsinogen from an unordered state to an intermediate state having ordered secondary structure. Interestingly, near-UV CD spectra show some amounts of stabilizing effect on the tertiary structure of the protein also. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on addition of EG and PEG. Maximum ANS binding occurs in presence of 80% EG and 90% PEG (v/v), suggesting the presence of an intermediate or molten globule-like state at high concentrations of the two polyols.     

Dual emission carbon dots from carotenoids: Converting a single emission to dual emission

Dual emission carbon dots have a high potential for use as fluorescence‐based sensors with higher selectivity and sensitivity. This study demonstrated the possibility of conversion of a biological molecular system with a single emission peak to a double emission carbon dots system. This report is the first to describe the synthesis of dual emission carbon dots by tuning the electronic environment of a conjugated system. Here we prepared carbon dots from a natural extract, from which carotenoids were used as a new source for carbon dots. Formation of the carbon dots was confirmed by images obtained under a transmission electron microscope as well as from a dynamic light scattering study. The prepared carbon dots system was characterized and its optical property was monitored. The study showed that, after irradiation with microwaves, the fluorescence intensity of the whole system changed, without any change in the original peak position of the carotenoid but with the appearance of an additional peak. A Fourier transform infrared study confirmed breaking of the conjugated system. When using ethylene glycol as a surface passivating agent added to these carotenoid carbon dots, the dual emission spectra became more distinct.     

Systematic investigation of interactions between papain and MPA-capped CdTe quantum dots

Fluorescent quantum dots (QDs) have been widely applied in biological and biomedical areas, but relatively little is known about the interaction of QDs with some natural enzymes. Herein, the interactions between 3-mercaptopropionic acid-capped CdTe QDs (MPA-QDs) and papain were systematically investigated by UV–Vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra under the physiological conditions. The fluorescence spectra results indicated that MPA-QDs quenched the fluorescence intensity of papain. The modified Stern–Volmer quenching constant K _a at different temperatures and the corresponding thermodynamic parameters ΔH, ΔG and ΔS were also calculated. The binding of MPA-QDs and papain is a result of the formation of QDs-papain complex and the electrostatic interactions play a major role in stabilizing the complex. The CD technique was further used to analyze the conformational changes of papain induced by MPA-QDs and the results indicated that the biological activity of papain was affected by MPA-QDs dramatically.     

Segregation of modified bacteriorhodopsin aggregations in reconstituted vesicle membrane induced by the change of thermodynamical parameters

It was clearly shown that the change in thermodynamical parameters could cause the segregation of membrane protein aggregations in the phospholipid membrane. At first, reconstituted vesicles were prepared with a membrane protein, bacteriorhodopsin and a constituent phospholipid of biomembranes, L-alpha-dimyristoyl phosphatidylcholine. When the temperature of the suspension was decreased or the osmotic pressure was increased by adding poly(ethylene glycol) to this vesicle suspension at 23 degrees, the circular dichroism spectra showed a typical band indicating bacteriorhodopsin trimer formation implying their aggregation. This suggests that the aggregation of trimers proceeded by adding poly(ethylene glycol) into vesicle suspension, just as it proceeded by decreasing the temperature. Next, vesicles were prepared with fluorescein isothiocyanate-labeled bacteriorhodopsin, photoemissive bacteriorhodopsin and L-alpha-dimyristoyl phosphatidylcholine. The excitation energy transfer between the two modified proteins was measured by fluorescence spectroscopy. In this case, however, when poly(ethylene glycol) was added into the suspension, the yield of the excitation energy transfer decreased. This result indicates that modified proteins aggregate separately in a segregated form in the vesicle membrane.     

Chemical modification studies of Artocarpus lakoocha lectin artocarpin.

The effect of chemical modification on an anti T-like lectin, artocarpin isolated from Artocarpus lakoocha seeds was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of carboxyl groups, arginine and lysine residues, did not affect the lectin activity. However, modification of tryptophan, tyrosine and histidine residues led to a complete loss of its activity, indicating the involvement of these amino acids in the saccharide-binding ability. A protection was observed in the presence of inhibitory sugar. A marked decrease in the fluorescence emission was found when the tryptophan residues of lectin were modified. The circular dichroism spectra showed the presence of an identical pattern of conformation in the native and modified lectin, indicating that the loss in activity was due to modification only. The effect of pronase on artocarpin showed loss of activity whereas papain and trypsin had no effect. The specific activity of artocarpin remained unaltered on treatment with glycosidases but remarkable increase in the activity (of the same) was observed with xylanase treatment. Immunodiffusion studies with chemically modified lectin showed no gross structural changes, indicating that the group specific modifying agents did not alter the antigenic sites of the modified lectin.     

Assay of ceftazidime and cefepime based on fluorescence quenching of carbon quantum dots

A novel and sensitive method for the determination of ceftazidime and cefepime in an active pharmaceutical ingredient (API) has been developed based on the fluorescence quenching of poly(ethylene glycol) (PEG)2000‐capped carbon quantum dots (CQDs) prepared using a chemical oxidation method. The quenching of fluorescence intensity is proportional to the concentration of ceftazidime and cefepime over the range of 0.33–3.30 and 0.24–2.40 µg/mL, respectively. The mode of interaction between PEG2000‐capped CQDs and ceftazidime/cefepime in aqueous solutions was investigated using a fluorescence, UV/Vis and Fourier transform infrared spectrometry (FTIR) at physiological pH. UV/Vis and FTIR spectra demonstrated that ground state compounds were formed through hydrophobic interaction the fluorescence quenching of CQDs caused by ceftazidime and cefepime. The quenching constants decreased with increases in temperature, which was consistent with static quenching. Copyright © 2015 John Wiley & Sons, Ltd.     

The thiol proteinases from the latex of Carica papaya L. I. Fractionation, purification and preliminary characterization

Three thiol proteinases, namely papain, chymopapain and proteinase omega were purified to homogeneity from the latex of Carica papaya L. During the purification procedure, the thiol function of the cysteinyl residues were protected either as mixed disulfides with cysteamine or 2-thiopyridone or as S-sulphenylthiosulfate derivative or after blocking with p-chloromercuribenzoic acid. In marked contrast with earlier publications, chymopapain also was found to be a monothiol proteinase as papain and proteinase omega. The active sites of chymopapain and proteinase omega could not be distinguished from that of papain neither by the analysis of the pH dependence of kcat/Km nor by the examination of the pH dependence of the fluorescence emission spectra.