黑布林籽β-葡萄糖苷酶聚合物的制备与稳定性研究

为探究β-葡萄糖苷酶聚合物的制备方法及其稳定性,以黑布林籽β-葡萄糖苷酶为原料制备交联酶聚合物(CLEAs),优化了制备过程中沉降剂的种类及比例、戊二醛交联剂的浓度等工艺参数,并采用扫描电镜分析其表面结构特征;在此基础上,以红景天苷的酶促合成为模型反应,研究CLEAs在有机溶剂中的热稳定性和操作稳定性。结果表明,制备β-葡萄糖苷酶CLEAs的最佳沉降剂为95%乙醇,其适宜添加量为酶液体积的3倍;戊二醛交联剂的最适浓度为30 mmol·L^-1,交联时间为1.0 h,酶活回收率高达91.4%。扫描电镜结果表明,在此条件下制备的CLEAs有较大的比表面积和致密的多孔道结构。稳定性结果表明,将β-葡萄糖苷酶CLEAs在60℃下保温2.0 h,其酶活回收率为75.1%,是游离酶的6.1倍;重复使用8次后,仍保持73.5%的初始酶活性。综上,黑布林籽β-葡萄糖苷酶CLEAs具有良好的生物催化活性、热稳定性和操作稳定性,可用于糖苷类化合物的高效合成。本研究为β-葡萄糖苷酶CLEAs的研究提供了理论依据,对工业化生产具有现实意义。     

高凝胶性蛋白粉制取工艺条件优化

为了进一步改善高凝胶蛋白粉的品质,探讨了利用酶交联反应制取高凝胶蛋白粉的新方法,在进行了琥珀酰化酪蛋白添加量、反应pH值、转谷氨酰胺酶添加量对蛋白粉凝胶强度影响的单因素试验基础上,该文通过响应面分析法对制取高凝胶性蛋白粉的酶交联反应条件进行了优化,并通过SDS-PAGE电泳、扫描电镜等手段探讨了酶交联蛋白粉凝胶性能增加的机理。结果表明,最佳工艺条件为:琥珀酰化酪蛋白添加量2.29%、反应pH值5.87、转谷氨酰胺酶添加量1.06%、反应温度40℃、反应时间40min。理论最大凝胶值为1072.8g/cm2,验证值为1054.5g/cm2,说明预测模型可靠性高,可用于反应条件的优化。     

不同因素对羔羊皱胃酶凝乳活性的影响

研究结果表明，羔羊皱胃酶最适凝乳温度为45℃；35℃以上热处理对酶凝乳活性有不同程度的损失，60℃热处理10 min活性完全丧失；pH值为5～8时凝乳活性随乳pH值的降低而增强，酶在pH2.5～7.5之间处理20h凝乳活性稳定；Ca²⁺具有明显的促凝作用；底物浓度对酶活性的影响符合米氏规律。     

碱酶两步法制备大米蛋白的研究

该文采用碱法和酶法两步加工碎米，制备得到纯度较高的大米蛋白。通过研究米粉粒度、pH值、温度、水料比和时间对提取率的影响，确定碱法制备大米蛋白的较佳工艺条件为：米粉粒度80目，pH 11.0，温度50℃，水料比8，时间120 min。采用α-淀粉酶对大米蛋白进行纯化，酶解条件为：加酶量60 U/g，pH 6.0，温度50℃，时间30 min。在上述工艺条件下制备大米蛋白，提取率为73.22%，纯度为88.75%。     

超高压处理对砀山梨汁中过氧化物酶活性的影响

该文研究了超高压处理对砀山梨汁中过氧化物酶活性的影响。试验压力为0.1～500 MPa，温度为20～60℃。此外，考察了不同pH值(3～7)和保压时间(2～34 min)超高压处理对酶活性的影响。试验结果表明：在处理温度为50℃、保压时间为10 min和梨汁pH 5的条件下，300 MPa以下压力范围内高压处理酶被激活，其活性增加；大于300 MPa时酶的活性随压力增大而下降。高压处理时，温度低于40℃对酶的活性影响不大；有效影响高压处理的最小温度为40℃。保压时间超过10 min后时间延长对酶的活性影响     

碳酸钙和壳聚糖联用对高pH值石灰性土壤砷污染的钝化

为明确碳酸钙-壳聚糖联用对高pH值石灰性土壤砷污染的钝化效果,该研究采用田间As污染模拟试验,设置对照(CK)、砷污染(As)、砷污染+碳酸钙(As+Ca)、砷污染+碳酸钙+壳聚糖(As+Ca+C)4个处理。研究了碳酸钙和壳聚糖添加对As污染石灰性土壤酶活性影响,污染土壤中As的形态变化,以及As在供试作物玉米体内的迁移特征。结果表明:As污染石灰性土壤上添加碳酸钙(Ca)有利于提高土壤酶活性,碳酸钙和壳聚糖(Ca+C)联用后,土壤脲酶、纤维素酶和过氧化氢酶活性分别显著提高了52.35%、74.92%、8.72%(P0.05)。高pH值石灰性土壤外源As的主要存在形态为残渣态,且残渣态占总砷含量的60%以上。与As处理相比,As+Ca和As+Ca+C处理的水溶态砷分别显著降低17.15%和27.03%(P0.05);As+Ca+C处理的钙-砷、铁-砷和铝-砷分别显著升高了13.97%、14.24%、13.85%(P0.05)。As+Ca和As+Ca+C钝化处理对As钝化率分别达9.78%和18.37%。As污染土壤上种植玉米会导致各部位As积累的增加,但Ca+C联用使玉米籽粒、根、茎、叶的As含量显著降低50%、13.98%、16.51%、14.94%(P0.05)。可见,Ca+C钝化剂联用方法可应用于高pH值石灰性土壤As污染修复。     

发酵鲭鱼中一株组胺降解菌的系统发育分析及特性研究

为筛选出具有优良组胺降解能力的细菌,并探索其在发酵食品中的应用,使用乳酸细菌(MRS)、结晶紫中性红胆盐葡萄糖琼脂(VRBDA)和2216E琼脂培养基对传统发酵鲭鱼(Scomber japonicus)中的组胺降解菌进行分离纯化,测定其生长、产酶特性及对发酵鲭鱼生物胺的降解作用。结果表明,菌株V-TB-2-5具有较高的降组胺活性,通过16S rRNA基因序列系统发育学分析结合形态特征确定为产氮假单胞菌(Pseudomonas azotoformans)。在营养肉汤培养基中,该菌的生长和产酶条件范围为20~40℃、pH值5~8、NaCl浓度0~3.5%(质量分数w/v),最适生长和产酶条件为30℃、pH值6、1.5%NaCl。酶反应的最适条件为30℃、pH值7。Mn²⁺、Mg²⁺和K⁺等金属离子对酶有激活作用,Fe³⁺或EDTA则有抑制作用。在传统发酵鲭鱼中,人工强化接种菌株V-TB-2-5与对照组相比组胺含量明显下降,组胺降解率为18.2%~40.7%;同时,接种组中亚精胺含量下降极显著,而精胺则上升。本研究为菌株V-TB-2-5在水产品加工和贮藏过程中组胺生物降解的控制提供了应用依据。     

葡萄糖基聚合物的酶催化合成及其在制备缓释微球中的应用

以脂肪酶Novozym-435催化葡萄糖和10- 十一碳烯酸合成了6-O-（10- 十一碳烯酸）-葡萄糖酯，在K2S2O8引发下合成了葡萄糖基聚合物。采用复凝聚法以葡萄糖基聚合物和海藻酸钠为基质材料，将非诺洛芬钙包裹制成缓释微球。通过L9（3^4）正交实验得出微球的最佳制备工艺条件，结果是葡萄糖基聚合物：海藻酸钠质量比=1：2，pH3.0，搅拌速度400r／min，反应成球温度45℃。在最佳工艺条件下制备的非诺洛芬钙缓释微球，粒径范围是10-20μm，平均药物包封率（73.74±3.12）％。同时，体外溶出试验表明，该微球具有较好的缓释作用。     

乳酸-乙醇酸共聚物/壳聚糖复式微球缓释蛋白

采用两次乳化包埋技术制备了一种载蛋白的乳酸-乙醇酸共聚物/壳聚糖复式微球（Poly（lactide-co-glycolide）/chitosan微球,简称PLGA/chitosan微球）。先以复乳法（W/O/W）制备加载牛血清白蛋白（BSA）的PLGA微球,再以壳聚糖（Chitosan）为基体对PLGA微球进行包埋,用三聚磷酸钠（TPP）进行交联。制备中,改变内部PLGA基体的分子量制备了3种PLGA/chitosan微球以达到不同的释放动力学。采用扫描电镜（SEM）、激光粒度分析仪、傅里叶变换红外光谱法（FTIR）分别对PLGA/chitosan微球的形貌、平均粒径及表面物理化学特征进行了表征。进行了复式微球在体外的蛋白释放实验,同时检测了体外降解过程中环境pH值的变化。结果表明,PLGA/chitosan微球具有球中包埋球的复式结构。复式微球的载药率为6%~8%,微球平均粒径为40~60μm。该微球早期蛋白的突释较PLGA微球显著减少,释放周期大于75天。此外,PLGA/chitosan微球降解过程中,能维持孵育液的pH值在7~8之间,为人体可接受范围,是一种优异的蛋白缓释微球。     

低温热处理对超高温灭菌乳中假单胞菌脂肪酶Ⅷ的活性影响

通过分析不同因素对假单胞菌脂肪酶Ⅷ活性的影响以及假单胞菌脂肪酶Ⅷ活性与超高温灭菌(UHT)乳脂肪上浮的关系，研究了控制UHT乳中假单胞菌脂肪酶Ⅷ活性和延缓脂肪上浮现象的措施。结果表明pH值对假单胞菌脂肪酶Ⅷ的活性有显著性影响(P<0.01)，50、55、60℃(20 min)的低温热处理能显著降低假单胞菌脂肪酶Ⅷ的活性(P<0.01)。贮藏试验表明低温热处理对控制UHT乳在贮存过程中的假单胞菌脂肪酶Ⅷ活性水平(P<0.01)和脂肪上浮现象(P<0.05)有明显的作用，而55℃和15 min的低温热处理对缓解UHT乳在贮藏过程中的脂肪上浮现象最为有效。     

beta-Lactoglobulin A with N-ethylmaleimide-modified sulfhydryl residue, polymerized through intermolecular disulfide bridge on heating in the presence of dithiothreitol

Roles of sulfhydryl groups on thermal aggregation of beta-lactoglobulin A (betaLG A) at pH 7.5 were investigated. It is known that betaLG A modified at Cys(121) with N-ethylmaleimide (NEM-betaLG A) does not form an aggregate by heating and that dithiothreitol (DTT) reduces cystine residues and induces the intermolecular sulfhydryl/disulfide interchange reaction and/or oxidation. NEM-betaLG A was heated in the presence of DTT. The molecules were linked together with an intermolecular disulfide bridge, and the polymer formed increased with increase in DTT concentration. The largest portion of polymer was formed when DTT was added at around the same molar concentration as that of NEM-betaLG A. Then, polymer formation decreased with further increase in DTT concentration. The results suggest that sulfhydryl/disulfide residues other than Cys(121), generated from cysteine residues, can induce intermolecular sulfhydryl/disulfide interchange reactions to polymer and that thiol compounds, for example, added DTT, are capable of starting such reactions.     

Study of the interaction of pancreatic lipase with procyanidins by optical and enzymatic methods

The interactions between porcine pancreatic lipase (PL) and grape seed procyanidins were studied by an enzymatic assay, fluorescence quenching, nephelometry, and dynamic light scattering (DLS). An inhibitory effect of grape seed procyanidins on lipase hydrolytic activity was found. Both the inhibition of lipase activity by procyanidins and the respective quenching of intrinsic protein fluorescence increased with the average degree of polymerization of the tested procyanidins. The association between procyanidins and enzyme involves a specific interaction as inferred from the fluorescence assays despite not changing significantly the tertiary structure of the protein. For all tested procyanidins it was shown, both by DLS and by nephelometry, that an increase in aggregation occurs up to a stoichiometric maximum after which further procyanidin addition causes a decrease in aggregation of aggregates. The maximum size of aggregates was shown to be closely related to the maximum overall aggregation. It was also shown that the inhibition of enzyme activity is to a large extent independent of the formation of aggregates.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

红壤交换性钙、镁和钾的分布及施肥对其影响

A leaching experiment was Carried out with repacked soil columns in laboratory to study the leaching process of a red soil derived from sandstone as affected by various fertilization practices.The treatments were CK(as a control),CaCO3,CaSO4,MgCO3,Ca(H2PO4)2,Urea,KCl,Multiple(a mixture of the above mentioned fertilizers) and KNO3,The fertilizers were added to the bare surface of the soil columns,and then the columns were leached with 120 mL deionized water daily through perstaltic pumps over a period of 92 days,At the end of leaching process,soils were sampled from different depths of the soil profiles ,i.o.,of 92 days,At the end of leaching process,soils were sampled from different depths of the soil profiles,I.e.0-5cm,5-10cm,10-20cm,20-40cm,and 40-60cm,The results showed when applying Ca,Mg,and K to the bare surface of the soil columns,exchangeable Ca^2 ,Mg^2 ,and K^ in the upper layer of the soil profile increased correspondingly,with an extent depending mainly on the application rates of Ca,Mg,and K and showing a downward trend,CaCO3,CaSO4,MgCO3,and Ca(H2PO4)2 treatments had scarcely and effect on movement of exchangeable K^ ,while CaCO3,and CaSO4 treatments singnificantly promoted the downward movement of exchangealble Mg^2 although these two treatments had no obvious effect on leaching losses of Mg,The fact that under Urea treatment,exchangeable Ca^2 and Mg^2 ,were higher as compared to CK treatment showed urea could prevent leaching of exchangeable Ca^2 and Mg^2 ,the obvious downward movement of exchangeable Ca^2 and Mg^2 was noticed in KCl treatment ,In Multiple treatment,the downward movement of exchangeable Ca^2 and Mg^2 was evident,while that of K^ was less evident,Application of KNO3 strongly promoted the downward movement of exchangeable Ca^2 and Mg^2 in the soil profile.     

黄土高原黄土团粒组成及其与碳酸钙关系的研究

通过团粒分析、电子显微镜 (SEM/EDX)扫描等元素分析扫描方法和手段 ,对黄土高原西北部自然黄土中的团粒数量及其与碳酸钙的溶解量的关系、团粒表面和内部碳酸钙的分布进行了研究。结果表明 :碳酸钙大约参与了 99%的团粒形成 ,呈现团粒越大 ,碳酸钙含量越多的趋势 ;反之 ,团粒数量与碳酸钙的溶解量成反比 ;随着团粒表面碳酸钙分布的减少 ,附着在团粒表面的微团粒及粉粒、粘粒减少 ;在团粒内部 ,碳酸钙与矿物颗粒之间存在 3种胶结方式 ,分别为碳酸钙附着在矿物颗粒表面、碳酸钙分布于矿物颗粒之间以及碳酸钙表面附着矿物颗粒。实验结果证明了黄土团粒的主要胶结物是碳酸钙这一理论。     

铁铝氧化物和高岭石对转化酶的吸附及活性的影响

Experiments were conducted to study the influences of synthetic bayerite, non-crystalline aluminum oxide (N-AlOH), goethite, non-crystalline iron oxide (N-FeOH) and kaolinite on the adsorption, activity, kinetics and thermal stability of invertase. Adsorption of invertase on iron, aluminum oxides fitted Langmuir equation. The amount of invertase held on the minerals followed the sequence kaolinite > goethite > N-AlOH > bayerite > N-FeOH. No correlation was found between enzyme adsorption and the specific surface area of minerals examined. The differences in the surface structure of minerals and the arrangement of enzymatic molecules on mineral surfaces led to the different capacities of minerals for enzyme adsorption. The adsorption of invertase on bayerite, N-AlOH, goethite, N-FeOH and kaolinite was differently affected by pH. The order for the activity of invertase adsorbed on minerals was N-FeOH > N-AlOH > bayerite > reak goethite > kaolinite. The inhibition effect of minerals on enzyme activity was kaolinite > crystalline oxides > non-crystalline oxides. The pH optimum of iron oxide- and aluminum oxide-invertase complexes was similar to that of free enzyme (pH 4.0), whereas the pH optimum of kaolinite-invertase complex was one pH unit higher than that of free enzyme. The affinity to substrate and the maximum reaction velocity as well as the thermal stability of combined invertase were lower than those of the free enzyme.     

Activite des complexes acides humiques-invertase: Influence du mode de preparation

During the preparation of insoluble humic acid-invertase-Ca²⁺ complexes, the humic acid, enzyme and Ca²⁺ concentrations were varied as well as the time of contact between the organic and the cation, and the order in which these three substances were added. The enzymatic activities of these complexes are very different. In complexes obtained at pH 7.0, adding humic acids first, then invertase and finally Ca²⁺, the enzyme molecules probably occur in two forms: some may be fixed on the surface, either by adsorption or covalence, the others may be trapped in a micellar net. The initial catalytic activity and lifetime of the complexes depends on the relative abundance of the three substances and on the time of contact. The kinetics of the first type of complex is divided into three parts. During the first very short period, a strong inactivation is noticed which can be explained in terms of three phenomena: separation of some micellae bound to enzyme molecules, discharge of trapped invertase molecules because of destructuring of the net after loss of micellae, desorption of the enzyme. The intermediate phase corresponds to a greater cohesion of the net since the loss of organic matter is reduced and the inactivation is slower; its length depends on the preparation. The final phase begins when the complex is free from labile fractions: it represents the residual activity of the complex and develops very slowly.If insoluble humate suspensions are used rather than humic acids solutions, the formation of humateinvertase complexes proceeds essentially by adsorption at the surface of the support. The kinetics of the catalytic activity of such enzymatic complexes is similar to that of desorption.     

生物质酶催化过程中pH值的非线性控制

为提高酶催化过程中pH值的控制精度,采用了具有参数自适应及Hammerstein模型2种相结合的控制策略方法,利用递推最小二乘进行参数估算,在控制器里颠倒静态非线性特性来对过程非线性部分进行补偿.此方式用于酶催化过程中的pH值过程控制,其试验结果不仅可使pH值高度非线性控制转换成一个近似的线性控制,并自动地改变控制器的整定参数,从而解决pH中和过程的高度非线性控制难点,还可达到提高酶催化过程中pH值的控制精度、提高酶的催化响应效果的目的.pH中和过程的计算机仿真结果表明,该控制算法比比例积分微分(proportion integral differential,PID)控制具有更好的控制品质.由于该控制主要是针对pH值的控制,有较强的通用性,具有典型应用价值和推广意义.     

Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent.

Among 10 lipases tested, Candida rugosa lipase exhibited the best ability to catalyze the resolution of dl-menthol in organic solvent. The lipase was immobilized on different carriers, and the experiment was carried out with different acyl donors. The high yield and optical purity of the product were achieved in cyclohexane with valeric acid as acyl donor using C. rugosa lipase immobilized on DEAE-Sephadex A-25. The conversion of dl-menthol depended on the water content of immobilized lipase and on the pH of the aqueous solution from which lipase was immobilized. The operational stability of the DEAE-Sephadex A-25 immobilized lipase in catalysis of the esterification reaction showed that >85% activity remained after 34 days of repeated use. The resolution of racemic menthol in organic medium catalyzed by immobilized C. rugosa lipase-catalyzed esterification is very convenient, and it represents a significant improvement in the use of enzyme for the preparative production of optically active menthol. This process is readily applicable to large-scale preparation.