Kinetic,thermodynamic parameters and in vitro digestion of tannase from Aspergillus tamarii URM 7115

Tannase is an enzyme used in various industries and produced by a large number of microorganisms. The aim of this study was to evaluate tannase production to determine the biochemical, kinetic, and thermodynamic properties and to simulate tannase in vitro digestion. The tannase-producing fungal strain was isolated from “jamun” leaves and identified as Aspergillus tamarii. Temperature at 26°C for 67?h was the best combination for maximum tannase activity (6.35-fold; initial activity in Plackett–Burman design—15.53?U/mL and average final activity in Doehlert design—98.68?U/mL). The crude extract of tannase was optimally active at 40°C, pH 5.5 and 6.5. Moreover, tannase was stimulated by Na⁺, Ca²⁺, Mg²⁺, and Mn²⁺. The half-life at 40°C lasted 247.55?min. The free energy of Gibbs, enthalpy, and entropy, at 40°C, was 81.47, 16.85, and ?0.21?kJ/mol?·?K, respectively. After total digestion, 123.95% of the original activity was retained. Results suggested that tannase from A. tamarii URM 7115 is an enzyme of interest for industrial applications, such as gallic acid production, additive for feed industry, and for beverage manufacturing, due to its catalytic and thermodynamic properties.     

Adaptive relationships of epoxide hydrolase in herbivorous arthropods

Epoxide hydrolase catalyzes a simple hydrolysis of reactive cyclic ethers that may otherwise alkylate and impair critical proteins and nucleic acids required for life. Although much less studied than the cytochrome P-450 monooxygenases that produce epoxides, differences in subcellular, tissue, pH, substrate, and inhibitor specificities argue for at least three forms of insect epoxide hydrolase. Increasing numbers of epoxides are being identified as plant allelochemicals, antifeedants, and essential hormones or precursors for herbivorous arthropods, and in many cases an associated alkene to diol pathway of metabolism is found. A role for epoxide hydrolase in arthropod-plant interactions is strongly supported by species comparisons and by age-activity and induction studies. Two major limitations for study in biochemical ecology of epoxide hydrolase are the lack of an effective in vivo inhibitor and a range of commercially available radiolabeled substrates for the enzymes.     

有机磷水解酶生物传感器载体固定化酶的研究

有机磷水解酶（OPH）传感器作为检测农产品中农药残留的新型检测装置,其酶的固定化对OPH传感器的灵敏度和稳定性有重要的影响。研究了几种酶固定化载体、孔径大小、固定方式、固定方法（试剂组成）对传感器pH值的影响。结果显示：采用孔径为0.45μm的硝酸纤维素膜制备固定化酶片的pH值要大于其余几种;采用浸泡方式制备固定化酶片的pH值明显大于传统的滴定法;采用牛血清白蛋白（BSA）、戊二醛交联固定的效果优于酶直接吸附法和BSA固定法,且当戊二醛体积分数为2.5％,BSA为10％时,酶固定化效果最好。     

盖子环替换及定点突变提高菜豆环氧化物水解酶对邻甲基苯基缩水甘油醚的催化性能

菜豆环氧化物水解酶1和2（PvEH1、PvEH2）能够动力学拆分外消旋邻甲基苯基缩水甘油醚（rac-oMGE）,从而保留(R)-oMGE。基于对PvEH1和PvEH2结构的同源模拟和分析,发现二者分子中的盖子环差异较大,故本文选择盖子环作为研究目标。经融合聚合酶链式反应（FPCR）,获得了PvEH2的盖子环区域被PvEH1对应区域替换的杂合酶Pv2Pv1。用全细胞酶E. coli/pv2pv1催化rac-oMGE,当(S)-oMGE刚好水解完全时,产物(S)-3-邻甲苯基-1,2-丙二醇（(S)-oTPD）的ee_p由PvEH2的58.3%提高至75.5%。为进一步提高酶的性质,在Pv2Pv1中选取11个氨基酸位点进行丙氨酸(A)突变,获得最优突变子E. coli/pv2pv1^K176A,活性为E. coli/pv2pv1（4.2U/g）的2.1倍,且当S构型的底物刚好完全水解时,(S)-oTPD的ee_p进一步提高为80.3%。分子对接分析发现,盖子环替换和K176位点突变为A,均使(R)-oMGE环氧环中的C_α更易受到酶中D101位点的攻击。利用E. coli/pv2pv1^K176A催化150mmol/L rac-oMGE水解制备(R)-oMGE（ee_s＞99%）和(S)-oTPD（ee_p=80.4%）,二者的产率Y_S和Y_P分别为32.7%和60.1%,时空产率STY_S和STY_P为1.6g/(L·h)和3.3g/(L·h)。本实验为改善EH的催化性质提供了一种有效策略。     

复合膳食纤维固体饮品的研制

以豆腐渣和海带为原料水解制备水溶性膳食纤维(SDF)和多糖,并制备复合膳食纤维固体饮品.用碱法和酶法结合提取大豆不溶性膳食纤维(IDF)和SDF,采用复合酶法制备海带多糖.结果表明:大豆IDF的提取条件为氢氧化钠浓度5%,浸泡时间60min、浸泡温度80℃;纤维素酶添加量为IDF重量的1%,纤维素与水的比例为1:12(W/V),pH为4.5,水解时间为12h,水解温度为40℃,在以上条件下,SDF的产率为36.01%.提取海带多糖条件为加入海带粉重1%的复合酶,复合酶(纤维素酶、果胶酶和蛋白酶)的最佳配比为2:1:2,在pH6.0、50℃酶解6h总多糖得率最高,可达10.71%.     

干酪乳杆菌GL-B体外降胆固醇的作用机理

研究了干酪乳杆菌GL-B所产胆盐水解酶的分离纯化及其体外降解胆固醇的作用机理,通过硫酸铵沉淀、DEAE-Sepharose FastFlow层析、Sephadex G-100层析分离胆盐水解酶。结果表明,胆盐水解酶纯度为原来的27倍,纯化后电泳谱图显示一条带,分子量约为50 ku;干酪乳杆菌GL-B对胆固醇的降解是吸收和共沉淀共同作用的结果,且共沉淀作用大于吸收作用。     

甲基对硫磷水解酶纯化与保存

对带有重组表达质粒E.coli菌种进行了扩大培养,在IPTG的诱导下大量表达,获得了C-末端含有6个寡聚组氨酸的甲基对硫磷水解酶。经Ni-NTA亲和层析获得了具有较高生物活性的甲基对硫磷水解酶。本文对该酶纯化的梯度洗脱条件和酶动力学进行了考察,并通过环境对酶活性的影响检测,确定了将酶制备成冻干粉,更利于保存。