Aspergillus ficuum菊粉酶的化学修饰

应用NBS、DEPC、EDC、DIC、Ch-T、PMSF 和DTT 化学修饰剂对Aspergillus ficuum 产内切菊粉酶和外切菊粉酶进行化学修饰,测定与其活性相关的氨基酸残基,结果表明,构成内切菊粉酶和外切菊粉酶活性中心的必需氨基酸残基均含有色氨酸和羧基氨基酸(谷氮酸或天门冬氨酸),组氨酸可能是酶活性中心的组成氨基酸。邹氏作图法进一步确认外切菊粉酶活性中心必需色氨酸残基数目为2,内切菊粉酶活性中心必需色氨酸残基数目为1 。     

Aspergillus ticuum菊粉酶酶学性质的研究

本实验对Aspergillus ticuum产菊粉酶体系中的外切菊粉酶和内切菊粉酶的酶学性质进行了研究,研究表明二者酶学性质非常相似:两种酶的最适反应温度均在45℃左右;外切菊粉酶的最适反应pH为4.5,内切菊粉酶为pH5.0; Ag 完全抑制两种酶的活性,Fe2 和Al3 对两种酶有较强的抑制作用,尤其是外切菊粉酶,Mg2 对两种酶有激活作用;实验还对两种酶的动力学常数进行了测定和比较.     

菊粉外切酶的异源表达、纯化及酶学性质

将菊粉降解菌Paenibacillus sp. Lfos16的菊粉外切酶基因克隆至表达载体p ET-28a (+),转入Escherichia. coli BL21(DE3)中实现了异源表达。利用镍柱亲和层析纯化重组菊粉外切酶,并进行SDS-PAGE检测。重组菊粉外切酶的表观分子质量为87 k Da,经纯化后,重组菊粉外切酶的比酶活为348.30 U/mg。重组菊粉外切酶作用菊粉和蔗糖的酶活分别为259.37 U/m L和592.16 U/m L,I/S值为0.438,且重组酶水解菊粉的主要产物为果糖。重组菊粉外切酶最适作用温度为40℃,且当温度低于30℃时酶活较稳定;最适作用pH为6。Ag~+、Cu~(2+)、Mn~(2+)、Zn~(2+)、Hg~(2+)、Fe~(3+)具有显著抑制作用。重组菊粉外切酶对菊粉的K_m为19. 28 mg/m L,Vmax为0.18 mg/(min·m L)。     

黑曲霉固体发酵产菊粉酶的研究

利用黑曲霉固体发酵获得了菊粉酶的粗酶液,并采用单因素试验和正交试验相结合的方法对酶的发酵条件进行了研究。结果表明,菊粉酶的最适发酵条件：pH值为6．5,培养6d,装样量为20g,氮源为酵母膏,基质为麦麸,可达到内切酶56．31U／g（干重）,外切酶137．24U／g（干重）。     

黑曲霉固体发酵产菊粉酶的研究

利用黑曲霉固体发酵获得了菊粉酶的粗酶液,并采用单因素试验和正交试验相结合的方法对酶的发酵条件进行了研究。结果表明,菊粉酶的最适发酵条件：pH值为6．5,培养6d,装样量为20g,氮源为酵母膏,基质为麦麸,可达到内切酶56．31U／g（干重）,外切酶137．24U／g（干重）。     

Aspergillus ficuum产果聚糖酶体系的分析

采用活性聚丙烯酰胺凝胶电泳法对Aspergillusficuum产果聚糖酶体系进行了分离 ,获得8条谱带 ;进一步运用薄层色谱 (TLC)和高效液相色谱 (HPLC)法进行分析 ,发现 8条谱带中有 3条属于外切菊粉酶 ,2条属于内切菊粉酶 ,证明了Aspergillusficuum能同时产内切菊粉酶和外切菊粉酶 .     

响应曲面法优化产外切菊粉酶菌株G-60的发酵条件研究

从新疆石河子盐碱地菊芋生长根际土壤中,分离筛选得到10株具有菊粉外切酶活力的菌株,复筛得到1株高产菊粉外切酶活力菌株,将其命名为G-60.在单因素实验的基础上,采用Plackett-Burman试验法对8个因素进行了显著因素的筛选,结果表明菊粉添加量、酵母膏含量和仞始pH值对菊粉外切酶酶活影响极显著;通过最陡爬坡试验及Box-Behnken设计进一步优化,得到最佳发酵产酶条件为:菊粉添加量7.91％、培养基初始pH为6.61、酵母膏含量0.64％,在此条件下,外切酶活达到58.51U/mL,与优化前相比提高2.02倍.     

发酵对酸肉蛋白质结构的影响

以猪背最长肌为原料制备酸肉,研究蛋白质结构在发酵中的变化。结果表明,随发酵时间延长,蛋白质静电作用力逐渐减弱,疏水相互作用和二硫键作用显著增强后呈小幅度波动变化。蛋白紫外吸收结果显示,肌原纤维蛋白芳香族氨基酸残基偏向更加疏水的环境移动,肌浆蛋白酪氨酸、色氨酸残基微环境极性增加。内源荧光图谱显示,荧光强度总体呈下降趋势且相对发酵0 d时发生红移,表明色氨酸发生了氧化降解,其残基主要暴露到极性环境中。拉曼光谱分析表明,随发酵进行,蛋白质α-螺旋减少,β-折叠增多,发酵110 d酸肉蛋白二级结构可能发生了重排现象。结果表明长时间发酵可使酸肉蛋白质二级结构和三级结构发生变化,这些变化可从蛋白质结构水平上解释发酵对酸肉品质的影响。     

黑曲霉原生质体诱变选育内切型菊粉酶生产菌株

从徐州市沛县河口镇秦庄村牛蒡种植基地腐烂牛蒡根附近采集土壤,经过初筛得到了3批菌株(包括从中国科学院购买菌株黑曲霉AS3.316),经过测定内切型菊粉酶酶活力(I)和外切型菊粉酶酶活力(S),筛选出I/S值大于10的菌株,共17株菌株。从17株黑曲霉菌株样本中,再筛选出一株产菊粉酶酶活力最高的菌株C122803,其酶活力为2.78U/mL。对菌株C122803进行原生质体制备及LiCl诱变后,得到一株内切型菊粉酶酶活力最高的菌株YY18,其酶活力为3.97U/mL,比出发菌株的酶活力提高了42.80%。     

黄酮“落新妇苷”与牛血清白蛋白相互作用研究

通过荧光法研究了落新妇苷与牛血清白蛋白(bovine serum albumin,BSA)之间的相互作用。考察了温度、BSA浓度、及常见离子对二者相互作用的影响。结果表明,落新妇苷具有较强的淬灭BSA内源荧光的能力,其淬灭机制为静态淬灭,表明二者可发生相互结合。计算了二者结合的热力学参数包括结合常数、焓变、熵变及自由能变化。结果表明,二者结合常数随温度的升高而缓慢增大,焓变及熵变均为正值说明疏水作用力在两者的相互作用中发挥重要的作用。Ca~(2+)、Mg~(2+)、K~+、SO_4~(2-)及NO_2~-等常见离子对落新妇苷与BSA相互作用的影响很小。同步荧光光谱研究表明落新妇苷会使BSA色氨酸残基附近疏水环境的极性增大。     

大豆蛋白肽-酪蛋白非磷酸肽组装产物的荧光光谱分析及抗氧化性研究

对不同p H值条件下制备的大豆蛋白肽-酪蛋白非磷酸肽组装产物(casein non-phosphopeptides-soybean peptide complex,CNPSPC)的荧光光谱及抗氧化性进行分析。结果表明,随着p H值增加,CNPSPC埋藏在疏水环境中的Trp残基暴露到分子表面的强度逐渐低于组装前的原料蛋白,且Trp残基所处的微环境极性有所降低。硫磺素T荧光光谱分析表明,p H 6.0的制备条件下,CNPSPC荧光强度最大,此时CNPSPC的β-折叠结构含量可能最多。组装改变了CNPSPC的Trp残基在空间结构中所处的微环境,使CNPSPC的分子构象发生改变。与酪蛋白非磷酸肽(casein non-phosphopeptides,CNPP)和大豆蛋白肽(soybean peptide,SP)相比,CNPSPC的抗氧化能力有所提高,在p H 6.0时其清除1,1-二苯基-2-三硝基苯肼自由基、O_2~-·、·OH能力达到最大,分别提高到组装前大豆蛋白的3.34、1.12倍和1.08倍。     

MTGase聚合酪蛋白酸钠生物聚合物的结构特征研究

采用紫外光谱、荧光光谱及红外光谱分析技术,研究了微生物转谷氨酰胺酶(MTGase)聚合酪蛋白酸钠(Na-CN)生物聚合物的空间结构特征,并探讨了MTGase改善Na-CN乳化性能的作用机理。紫外光谱显示,MTGase聚合Na-CN生物聚合物的多肽链的Trp和Tyr残基的紫外吸收峰的强度明显低于Na-CN,说明生物聚合物的“空间结构效应”占较重要的地位。荧光发射光谱显示,Na-CN生物聚合物的Trp和Tyr残基的荧光强度比Na-CN有显著的增强,表明生物聚合物的疏水性区域更加暴露。然而,MTGase长时间催化(12h)得到的生物聚合物的荧光强度反而有所下降(与4h的场合相比),这反映了“空间位阻效应”。红外光谱显示,Na-CN与其生物聚合物的酰胺特征峰相差不大,说明两者的二级结构基本上相近。此外,MTGase改善Na-CN乳化性能的机理是:MTGase催化导致Na-CN的空间结构发生了变化,进而改变了蛋白表面的表面疏水性质,最终达到改善Na-CN乳化性质的效果。     

Binding of curcumin to β-lactoglobulin and its effect on antioxidant characteristics of curcumin

The binding of curcumin (CCM) to bovine β-lactoglobulin (β-Lg) was investigated by Fourier transform infrared and fluorescence. The effect of binding on antioxidant activity of CCM was determined by using ABTS and hydroxyl radical scavenging capacity and total reducing ability. Our results showed that when CCM binds to β-Lg, it lead to a partial change in protein structure. In fact, CCM was bound respectively to two different sites of protein at pH 6.0 and 7.0 via hydrophobic interaction. CCM–β-Lg complex was formed by one molecule of protein combining with one molecule of CCM. Moreover, the average distance from one binding site to Trp residues in protein is similar with another. This result suggested that fluorescence resonance energy transfer cannot be used as unique method to study the characteristics of binding of ligands to proteins. The antioxidant activity of CCM might be improved by binding with β-Lg.     

Hydrophobic interactions in human casein micelle formation: beta-casein aggregation

The association of non-phosphorylated (0-P) and fully phosphorylated (5-P) human beta-caseins was studied by fluorescence spectroscopy and laser light scattering. The tryptophan fluorescence intensity (FI) level increased between 20 and 35 degrees C, indicating a change in the environment of that residue. A similar transition occurred when ANS was used as a probe. Transition temperatures were slightly lower in 10 mM-CaCl2 but were not affected by an equivalent increase in ionic strength caused by NaCl. The magnitude of the FI change was less for the 5-P than the 0-P protein but was increased for both by CaCl2 addition. These FI data were characteristic of a conformational change and this was supported by fluorescence polarization which indicated that with CaCl2, tryptophan and ANS mobility increased at the transition temperature even though the extent of protein association also increased. Light scattering suggested that protein association proceeded with the primary formation of submicellar aggregates containing 20-30 monomers which then associated further to form particles of minimum micelle size (12-15 submicelles), and eventually larger. The temperature of precipitation of the 5-P form in the presence of CaCl2 was lower than the conformational transition and suggested that both hydrophobic interactions and Ca bridges between phosphate esters on adjacent molecules are important in micelle formation.     

云南小粒咖啡类黑精荧光矩阵光谱特征

本文以云南小粒咖啡为原料制备类黑精,采用三维荧光矩阵光谱分析技术（Three dimensional excitation emission matrix spectra, 3D-EEMs）研究咖啡类黑精在不同温度、不同pH、不同光照及存放时间、不同浓度条件下的荧光光谱特征变化。结果表明:在10～80 ℃范围内云南小粒咖啡类黑精水溶液荧光强度与温度成反比;pH介于5～10之间的类黑精水溶液荧光强度较为稳定;避光存储的类黑精水溶液在60 min内荧光强度变化不显著（P>0.05）,而光照时间增加会导致荧光强度显著下降（P<0.05）;在浓度小于0.5 mg/mL时,类黑精水溶液荧光强度与浓度成正比,相关系数为0.9846;浓度大于10 mg/mL时会发生荧光猝灭,类黑精水溶液荧光强度与浓度不相关。本研究将为咖啡类黑精光谱特征的深入研究提供一定数据支持。     

Study on β-lactoglobulin glycosylation with dextran: effect on solubility and heat stability

In this paper, the characteristics of purified conjugates formed between β-lactoglobulin (β-lg) and a high molecular weight dextran under different dry heating conditions were investigated. SDS–PAGE analyses under non-reducing and reducing conditions showed that glycation of β-lg led to the formation of high molecular weight complexes and induced polymerization of the protein by disulfide bonds. The fluorescence emission spectra did not show changes in λ_max, which was indicative of a similar conformation around Trp residues. The conjugate formed at 60 °C, 0.44 a_w and 2:1 weight ratio of dextran to β-lg (conjugate 1) exhibited a fluorescence intensity very similar to that of the native protein and was selected to study the influence of glycosylation on the solubility and heat-stability properties. Solubility of conjugate 1 was higher than that of the dry-heated β-lg in the pH range from 3 to 9 and, particularly, around the isoelectric point of the protein. As compared to the native protein, the solubility of the conjugate decreased at pH 4. The glycated β-lg presented lower stability to heating at pH 7.0 than native β-lg, but its thermal stability was higher at pH 5.0 at temperatures above 85 °C.     

Fluorescence and coloration of grey hair

Grey hair samples were collected from 11 individuals and separated into un‐pigmented and pigmented fibres (International Hair Importers). Fluorescence measurements were obtained by using a double‐grating fluorescence spectrophotometer and a bifurcated fibre optics accessory to measure the spectra directly from the surface of hair at various distances from the fibre root. Colour measurements were carried out by using a Hunter colorimeter. The fluorescence spectra of un‐pigmented hair obtained by the excitation at 290 nm show a peak at 356 nm [tryptophan (Trp)], and multi‐peak emissions in the range from 395 to 500 nm. A significant variation in the Trp emission intensity at 356 nm vs. the intensity of emission in the 395–500 nm range was observed for hair collected from various individuals with yellow coloured hair producing stronger relative emission in 395–500 nm range. Quantitative measurements of coloration and the calculation of the Yellowness Index (YI) showed linear correlation between YI and the ratio of fluorescence intensities I₄₄₀/I₃₅₆ The spectra obtained by excitation at 320 nm showed the emission peaks at 395 nm (unidentified), 420 nm (N‐formylkynurenine), 460 nm (kynurenine), and 495 nm (3‐hydroxykynurenine), which are the products of oxidative or metabolic conversion of tryptophan. Un‐pigmented, yellow hair showed a build‐up of the fluorescence band corresponding to 3‐hydroxykynurenine at 495 nm. The data also showed the fluorescence quenching effect of melanin resulting in the lowering of the fluorescence intensity of pigmented hair. The spectra obtained at various positions along the fibres demonstrated gradual photo‐decomposition of hair chromophores during their lifetimes. This was indicated by a decrease of Trp fluorescence intensity, which was relatively fast (8·10^?4–1.5·10^?3 [day^?1] as calculated for hair obtained from various individuals) for un‐pigmented hair and slower for pigmented hair. A decrease in Trp emission was accompanied by an increase in the yellow coloration toward the ends of un‐pigmented fibres.     

曲霉SK004产菊粉酶发酵条件的确定及酶学性质研究

对实验室保存的 1株代号为SK0 0 4无花果曲霉菌株进行了发酵条件的优化 ,确定该菌株产胞外菊粉酶 ,且主要为外切菊粉酶。优化条件为 (g /L) :菊粉 2 5 ,蛋白胨 2 5 ,NH4H2 PO44,NaCl 5 ,MgSO4·7H2 O 0 5 ,Zn SO4·7H2 O 0 1,pH 6 5 ,30℃振荡培养 7 5d ,酶活力达到 5 3 1U/mL。酶反应的最适pH为 4 5 ,最适温度 6 0℃ ,在 pH 3 0～ 8 0的范围内和 6 0℃以下保存时具有较好的稳定性。     

Fluorescence inner-filtering correction for determining the humification index of dissolved organic matter

The use of fluorescence spectrometry has been suggested as a simple method to determine the extent of natural organic matter humification by quantifying the red-shifting of fluorescence emission that occurs with increasing humification. Humification indices are calculated by dividing fluorescence intensity at longer wavelengths by intensity at shorter wavelengths. These indices calculated without any specific efforts to standardize dissolved organic matter (DOM) concentration will result in index values thatvary with DOM concentration due to fluorescence innerfiltering effects. This study critically evaluated the effect of DOM concentration on humification index determination using organic matter isolated from field corn extract, soil: water extract, and soil fulvic acid. The results show that humification index values are sensitive to DOM concentration of the solution and are linear with respect to transmittance of the solution at the 254 nm used as the excitation wavelength. An approximate correction for DOM is to exploit the linear nature of the regression fit and to determine index values at the extrapolated 100% transmittance value. An exact correction using explicit correction factors for both primary and secondary innerfiltration effects was shown to give humification index values that are concentration invariant when absorbance of the solution at 254 nm was less than approximately 0.3 unit. Defining the humification index as the fluorescence intensity in the 300-345 nm region divided by the sum of intensity in the 300-345 nm and 435-480 nm regions was statistically advantageous. This study suggests that for quantitative results which can be used to compare humification of natural organic matter across different studies, correction of the fluorescence emission spectra for innerfiltration effects is needed.