Nocardia sp.腈水合酶的纯化过程研究

对Nocardiasp.高活力腈水合酶进行了纯化研究。在细胞破碎中,超声时间对腈水合酶比酶活存在一个最优值,超声时间为20.00min时得到的比酶活最高。在离子交换层析过程中,采用DEAE-Sepharose作为层析介质,分别对平衡缓冲液pH、离子强度和线性梯度洗脱体积进行了优化。结果表明,采用pH7.20、50mmolL-1的Na2HPO4-NaH2PO4溶液作为平衡缓冲液,0.00~1.00molL-1的NaCl线性梯度洗脱,洗脱体积为20~25倍柱体积,此条件下腈水合酶的纯化倍数和酶活收率较佳。以Phenyl-SepharoseFF为层析介质研究了疏水层析精制腈水合酶的工艺过程,采用两步层析优化方法纯化出的Nocardiasp.腈水合酶比酶活达到2648.0U穖g-1,酶活收率为40.84%,用SDS-PAGE检测其纯度为99.00%以上。Nocardiasp.腈水合酶的两个亚基分子量分别为22.90kDa和27.38kDa。     

仙人掌超氧化物歧化酶的纯化及其部分性质研究

10 0 0g仙人掌茎经匀浆、硫酸铵分级沉淀、SephadexG - 75凝胶层析和DEAE - 5 2离子交换层析 3个步骤 ,将仙人掌SOD纯化到SDS -PAGE均一纯 ,得白色粉状SOD 0 8985g ,比活 910U/mg,活力回收 4 3 1% ,纯化倍数 76。该酶相对分子质量 37 1kD ,H2 O2 和KCN的抑制实验结果表明为Cu、Zn -SOD。该酶的最大紫外吸收在 2 75nm处。 5 0℃保温 15 0min ,SOD的活性不变 ,在pH =5 0～ 9 0时SOD有较好的稳定性     

植酸酶的激光诱变改性与分离纯化

植酸酶产生菌———黑曲霉(Aspergillus niger)可在不同的辐照条件下进行激光诱变,确定了适宜的诱变条件,即输出波长为1.06μm的Nd:YAG高重复率声光调Q激光,辐照频率4 kHz,功率10 W,水平照射2~2.5m in,并建立了变异菌株基因库。通过高通量筛选,从中筛选到一株酶学性质比较符合工业生产要求的变异菌株phy226,pH=2~3的第二酶活峰提高了40%,酶活温度稳定在30~60℃。发酵液经盐析、透析、凝胶层析和离子交换层析等分离纯化过程,最终纯化倍数达7.8,收率为25.2%。     

大豆脂肪氧合酶(LOX)的固定化及增强稳定性

以活性白土、滑石粉为载体采用吸附法固定化大豆脂肪氧合酶(LOX)的优化条件为:(1)酶液对活性白土的用量比为897U/mg,在浓度为0 05mol/L、pH=7 0的磷酸盐缓冲液中20℃搅拌吸附30min;(2)酶液对滑石粉的用量比为238U/mg,在浓度为0 05mol/L、pH=8 0的磷酸盐缓冲液中10℃搅拌吸附15min。经放大后实验重复性良好。上述固定化酶置于0～4℃保存20d,酶活损失分别为19 2%和17 7%;与游离酶相比,能加快酶促反应的速度并使产率分别提高9 6%和42 5%。以海藻酸钠为载体采用包埋法得到的固定化酶珠的耐热、耐酸碱、耐有机溶剂能力较游离酶有很大提高,在φ(甘油)=75%的水溶液中保存33d,酶活基本保持不变;在50℃热处理60min后酶活仍能保持90%。     

壳聚糖凝胶吸附蛋白质机理研究

以壳聚糖为原料,经固化、交联、还原的工艺路线,制备了一种层析凝胶,对该凝胶吸附机理进行了研究。动力学研究表明,在初始ρ(牛血清蛋白)=1mg/mL和pH=6 0时,凝胶对牛血清蛋白的吸附在30min内可达平衡,按Lagergren方程计算出一级速率常数为15 63h-1,吸附平衡的实验数据符合Langmuir和Freundlich关系式,红外光谱测定表明,凝胶中与蛋白质或酶离子的相互作用主要是以氢键形式存在的静电引力。     

耐酸性高温α-淀粉酶突变基因的异源表达及纯化

将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上,实现重组质粒pET-amyd在大肠杆菌BL21(DE3)中的高效表达.经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析,重组酶AMYD的比活达到354.6 U·mg-1、纯化倍数为83.83,获得凝胶电泳条带单一的蛋白样品,经SDS-PAGE检测,AMYD酶分子量为63.5 kDa.重组酶AMYD的最适温度80℃、最适反应pH值为4.5,在温度低于90℃、反应pH值4.0～6.5的条件下,酶活较稳定.     

蛋清中溶菌酶的提取研究

以鸡蛋清为原料 ,控制温度 5℃ ,采用 72 4型树脂吸附 ;再用 φ(H2 SO4 ) =1%的稀硫酸分四次洗脱 ,用NaOH调pH =8 5～ 9 0 ,除去杂质 ;再用c(HCl) =3 0mol/L的盐酸调pH =3 5 ,加入w (NaCl) =5 %的溶液使溶菌酶析出 ,用无水丙酮 0℃干燥即得溶菌酶成品。     

基于疫苗颗粒完整性的硅胶吸附/解吸附纯化重组乙肝表面抗原 工艺研究

针对重组汉逊酵母乙肝表面抗原(HBsAg)在传统纯化过程中稳定性差的问题，采用静态光散射、荧光光谱和动态光散射等分析手段，从颗粒完整性角度研究其在不同pH条件下的稳定性变化。以其为指导，采用硅胶吸附/解吸附纯化HBsAg，建立该过程中关键因素的响应面模型，并与疏水层析联用，进一步纯化HBsAg，分析纯化效果以及纯化后疫苗颗粒完整性。采用透射电镜观察纯化后疫苗形貌，用高效分子排阻色谱法(HPSEC)分析颗粒稳定性。结果表明在酸性溶液下，pH接近HBsAg等电点时，抗原颗粒间静电斥力减小，颗粒容易聚集；碱性条件下，抗原颗粒内部疏水基团暴露，造成颗粒解聚。建立响应面模型，以活性收率为响应值时，最佳纯化工艺为吸附pH=7.43，洗脱pH=10.48，洗脱温度55.4℃，此时活性收率最高为39.1%；以纯化倍数为响应值时，吸附pH=7.16，洗脱pH=10.52，洗脱温度55.1℃，此时纯化倍数最高为1.90。进一步对洗脱液进行疏水层析纯化，活性收率为49.73%，颗粒完整性为85.79%，透射电镜观察到抗原颗粒粒径为20~40 nm。与传统疏水层析方法相比，采用硅胶吸附/解吸附与疏水层析联用的纯化方法，疫苗活性收率提高31.99个百分点，颗粒完整性提高20.90个百分点，颗粒稳定性提高22.93个百分点。该研究为高效纯化重组HBsAg及提高疫苗纯化过程的颗粒完整程度等提供新思路。     

一种竹腐朽菌胞外木聚糖酶的分离和特性研究

从腐朽的竹子上分离得到一株产木聚糖酶侧耳真菌Pleurotus sp.GH196,研究了其液体振荡培养的产酶条件。适宜碳源是1%稻草粉与1%麸皮组成的复合碳源,氮源是0.2%NH4NO3。经过硫酸铵分级沉淀、DEAE-Sephadex A-25离子交换层析、Sephadex G-100凝胶过滤三步纯化得到木聚糖酶的一个主要酶活组分A。该木聚糖酶在40～50℃、pH 4.0～5.5可以保持较好的稳定性,酶活最适反应条件是温度55℃、pH 4.5。     

核酸酶P1的纯化和酶学性质研究

桔青霉发酵液通过硫酸铵分级沉淀、凝胶层析和离子交换纤维素层析等生化分离步骤,得到的核酸酶P1比活力为711U/mg,纯化倍数1500。纯化的酶蛋白纯品经SDS 聚丙烯酰胺电泳,呈现一条单带。该酶催化反应的最适pH范围为6～8,最适温度在70℃,在60℃以下时酶活较为稳定.锌离子对此酶有明显的激活作用,而铜离子具有明显的抑制作用。     

Optimization of elution conditions of chitosan gel and the study of its adsorbent mechanism

Chitosan gel was prepared by crosslinking method. To make use of chitosan gel, the optimization of elution conditions and the adsorbent characteristics of chitosan gel were discussed in this article. The optimum elution conditions were c (NaCl) = 0.05 mol/L in Tris‐HCl (pH, 9.05) at the flow rate of 2.0–3.0 mL/min with chitosan gel (particle sizes, 120–140 μm). The effects of contact time, pH, initial BSA concentration, and temperature on adsorption were studied. The equilibrium data could be described well by Langmuir, Freundlich, and Redlich–Peterson models. Adsorption dynamics had been successfully studied by Langergren, intraparticle diffusion model and Avrami model. The thermodynamics parameters ΔG°, ΔH°, and ΔS° were calculated. The spectra studies indicated that the interaction between gel and protein was chiefly through electrostatic attraction in the shape of hydrogen bond. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1495–1506, 2007     

Swelling Properties of a Nickel-containing Cross-linked Chitosan

The swelling kinetics and equilibrium swelling degree of a series of nickel-containing chitosan cross-linked with glutaraldehyde at amino:aldehyde reactant ratios of 1:0.05, 1:0.17, and 1:0.5 were studied. At an amino:aldehyde reactant ratio of 1:0.17 the cross-linked chitosan was shown to swell in the basic solvent (50 mM tris-HCl, 0.2 M NaCl, pH = 7.5) and to have the best nickel retaining properties. Based on these results, the nickel-containing cross-linked chitosan has been concluded to be a potentially good candidate for the specific binding of proteins having terminal histidines.     

聚氧化乙烯/海藻酸钠/羟丙基-癸烷基壳聚糖网络凝胶的制备及其对三七总皂苷的体外释放性能研究

将壳聚糖改性成两亲性的羟丙基-癸烷基壳聚糖,并通过化学交联方法制备了聚氧化乙烯/海藻酸钠/羟丙基-癸烷基壳聚糖载药网络凝胶。利用傅利叶变换红外光谱仪、X-射线衍射仪、扫描电子显微镜对凝胶结构进行了表征。实验结果表明,该凝胶具有良好的网状结构和溶胀性,同时兼具p H敏感性和温度敏感性,在不同p H环境中均对三七总皂苷具有一定的缓释作用。     

HPLC测定盐酸格拉司琼葡萄糖注射液中盐酸格拉司琼含量及有关物质

采用氰基硅烷键合硅胶柱(250 mm×4.6 mm,5μm),以0.05 mol/L醋酸钠缓冲液[含0.25%(mL/mL)三乙胺,用冰醋酸调节pH值至6.0]-甲醇(50∶50)为流动相;在0.8 mL/min的流速和302 nm的检测波长下用高效液相色谱方法测定盐酸格拉司琼葡萄糖注射液中盐酸格拉司琼含量及有关物质。结果表明,盐酸格拉司琼在19.8～46.2μg/mL浓度范围内呈现出良好线性关系,线性相关系数为0.999 6,平均回收率是98.18%,RSD是1.32%,且5-羟甲基糠醛在4～14μg/mL浓度范围内呈现出良好线性关系,线性相关系数为0.999 8,检出限为1ng。同时,各杂质均可与盐酸格拉司琼主峰良好分离。该方法简便、快速、准确、专属性强及灵敏度高。     

Gel based on modified chitosan for oil spill cleanup

A method has been developed for obtaining a polymer porous gel and coating based on nontoxic and environmentally friendly components: chitosan, citral, and glutaraldehyde. The chemical structure and morphology of the polymer gel were investigated by infrared spectroscopy and scanning electron microscopy, respectively. The contact angles were determined using a goniometer, and the water and oil absorption of the gel were determined by the gravimetric method based on changes in the gel mass. Measurement of the contact angle θ of transmission oil and water drops on the coating surface showed the hydrophobic nature of the coating (θ = 90° for water and θ = 0° for oil). Study of the sorption properties of the resulting gel showed high sorption capacity with respect to transmission oil (9.05 kg/kg) and low sorption capacity with respect to water (2.46 kg/kg). It was found that after oil desorption from the loaded aerogel, it can be reused. The potential possibility of recycling the spent gel through its biodegradation in the soil was shown. Because of the excellent sorption capacity, high porosity, low density, and soil degradability, the developed gel has a great potential for application in the field of environmental purification from oil pollution.     

Development of ultrafine chitosan fibers through modified wetspinning technique

Chitosan has been extensively exploited in biomaterials research because of easy tailorable properties. Chitosan fibers are produced through either wetspinning or electrospinning. However, it is difficult to produce few microns fibers using either of these techniques. Present study focuses on production of ultrafine chitosan fibers through modified wetspinning technique by injecting homogenous chitosan solution through a very fine hole of silicone tube into either sodium tripolyphosphate (STPP) or sodium hydroxide (NaOH) bath by applying positive pressure. The gelation behavior of the chitosan was evaluated with STPP and NaOH solution through rheological study for comparative spinnability of chitosan in STPP and NaOH bath. Although gel strength of chitosan–NaOH system (240 Pa) was four times higher than that of chitosan–STPP system, gel breakdown rate was higher in previous case. From Fourier infrared spectroscopy (FTIR) analysis, ionic cross‐linking between TPP and chitosan molecules in chitosan–TPP fibers was confirmed. Scanning electron micrographs showed fine chitosan fibers with average diameter of ～ 10 μm. These nonwoven fibers/scaffolds with interconnected porosity may find potential biomedical applications. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

磁性壳聚糖微球的合成及其在固定化血管紧张素转化酶的应用

以化学共沉淀法合成Fe3O4纳米粒子为磁核,采用乳化交联法制备磁性壳聚糖微球,并对其形貌、结构和磁饱和强度等性质进行了表征。以磁性壳聚糖微球作为载体,固定化猪肺粗提物中的血管紧张素转化酶,并对固定化条件进行研究。结果表明,固定化血管紧张素转化酶的最佳条件为：pH值为8.3,最佳温度为50 ℃,最佳时间为1.5 h,最佳酶溶液蛋白浓度为6 mg/mL,此时固定化酶活力最高为0.048 U/g微球。与游离酶相比,固定化酶的pH值稳定性和热稳定性均得到提高。固定化酶重复使用10次,仍然保持40%以上相对活力,说明磁性壳聚糖微球是固定化血管紧张素转化酶的良好载体。     

反相高效液相色谱法同时测定乳酸及乳酸甲酯

乳酸及乳酸甲酯是重要的有机化工原料。在由乳酸合成乳酸甲酯的绿色化工工艺研究过程中,为了优化反应条件,建立了一种同时分离测定乳酸连续酯化反应体系中乳酸和乳酸甲酯的反相高效液相色谱法。HPLC分析条件为:Prevail C18色谱柱,以V〔NaH2PO4/H3PO4缓冲液(pH2.0)〕:V(甲醇)=90∶10为流动相,流速为1.0 mL.m in-1,检测器为示差折光检测器。在进样量为0.2~20μg成良好的线性关系。乳酸的加样回收率为94.55%~106.94%,相对标准偏差为1.23%~2.01%;乳酸甲酯的加样回收率为91.03%~109.0%,相对标准偏差为1.25%~2.88%。该方法准确、简捷、可靠。     

高效液相色谱法分析复配除草剂中苄嘧磺隆和丁草胺

采用反相C18柱和紫外检测器 ,对丁·苄可湿性粉剂中苄嘧磺隆和丁草胺的测定方法进行了研究。结果表明 ,以甲醇 (A)和醋酸 -醋酸钠缓冲溶液 (B ,pH =5 .6 )为流动相 ,采用梯度淋洗 :0～ 5 .5min ,A∶B =6 5∶35 (v/v) ,5 .6min后 ,改为A∶B =85∶15 (v/v) ;在波长2 5 4nm下检测 ,苄嘧磺隆和丁草胺的变异系数分别为 1.2 0 %和 0 .83% ,平均回收率分别为99.4 %和 99 9% ,苄嘧磺隆和丁草胺的浓度分别在 0 0 0 1～ 0 36 0mg/mL和 0 0 15～7 74 0mg/mL的范围内线性关系良好。方法准确、快速、重现性好 ,已用于丁·苄可湿性粉剂的质量控制。