葡萄糖氧化酶-过氧化物酶偶联体系葡萄糖分析

酶催化反应具有高效、快速、高选择性,在定量分析领域应用广泛.本文采用葡萄糖氧化酶-过氧化物酶偶联体系,用动力学方法对样品中葡萄糖含量进行分光光度测定,得到了较为满意的结果.     

Biochemical Characterization of Pyranose Oxidase from Streptomyces canus—Towards a Better Understanding of Pyranose Oxidase Homologues in Bacteria

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.     

Properties,Physiological Functions and Involvement of Basidiomycetous Alcohol Oxidase in Wood Degradation

Extensive research efforts have been devoted to describing yeast alcohol oxidase (AO) and its promoter region, which is vastly applied in studies of heterologous gene expression. However, little is known about basidiomycetous AO and its physiological role in wood degradation. This review describes several alcohol oxidases from both white and brown rot fungi, highlighting their physicochemical and kinetic properties. Moreover, the review presents a detailed analysis of available AO-encoding gene promoter regions in basidiomycetous fungi with a discussion of the manipulations of culture conditions in relation to the modification of alcohol oxidase gene expression and changes in enzyme production. The analysis of reactions catalyzed by lignin-modifying enzymes (LME) and certain lignin auxiliary enzymes (LDA) elucidated the possible involvement of alcohol oxidase in the degradation of derivatives of this polymer. Combined data on lignin degradation pathways suggest that basidiomycetous AO is important in secondary reactions during lignin decomposition by wood degrading fungi. With numerous alcoholic substrates, the enzyme is probably engaged in a variety of catalytic reactions leading to the detoxification of compounds produced in lignin degradation processes and their utilization as a carbon source by fungal mycelium.     

游离胆备醇酶传感器的研制

本文初步研制了一种测定游离胆固醇的酶传感器。该传感器用于流动注射法的最佳工作条件:磷酸盐缓冲液作载液(pH=6.3,0.1mol/L),流动速度为0.5mL/min,温度为27℃,检测限为0.15mg/mL～1.14mg/mL,线性响应范围0.25～1.00mg/mL,斜率0.08μA·mL/mg,响应时间为0.5～2.0min,工作寿命15d。已将该传感器用于样品分析,取得初步满意的结果。     

用于血糖无创检测的葡萄糖传感器研究

研制一种新型葡萄糖传感器,初步实现用于反离子电渗透技术提取皮下组织液的葡萄糖的浓度测量。用铁氰化钾作为电子媒介体,固定在聚环氧乙烷凝胶里的葡萄糖氧化酶与溶液中的葡萄糖催化氧化生成葡萄糖酸和亚铁氰化钾,通过检测该反应产生的氧化还原电流的大小来计算葡萄糖溶液的浓度。新型葡萄糖传感器的检测浓度范围2～22 mmol/L内线性度较好,传感器的一致性测试表明:同一传感器多次测量的偏差不超过2%,反应时间较短,接近于1 s。     

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.     

外源过氧化氢酶对液态发酵红茶色素的影响

为得到优质速溶红茶,本研究以低档绿茶为原料,对其水提液添加外源过氧化氢酶(CAT)进行液态发酵酶促转化成红茶茶汤,探讨液态发酵工艺对红茶色素生成量的影响,结果表明:参照优质红茶色素含量标准,CAT酶促转化的最优条件为:加酶量0.25 U/m L,反应温度45℃,反应时间2 h,p H6。CAT与多酚氧化酶(PPO)存在双酶协同作用,绿茶水提液先用PPO处理后(酶添加量0.05 U/m L,温度40℃,p H5,时间1.5 h)再用CAT处理,合成红茶色素的效果比单一酶源好。      

预浸泡对桃金娘果汁中抗氧化物质和抗氧化活性的影响

本研究将桃金娘整果分别浸泡在不同温度的水、盐酸、柠檬酸、抗坏血酸和亚硫酸氢钠溶液中10、20、30 min,分析不同处理对桃金娘果汁中总多酚、总花青素、总黄酮和抗氧化活性的影响。结果表明,在40⁶0℃水中浸泡,桃金娘果汁中总多酚、总花青素、总黄酮含量和抗氧化活性显著降低,然而在80℃热水和1%盐酸中预浸泡可保持或增加抗氧化物含量。柠檬酸、抗坏血酸或NaHSO₃溶液能降低抗氧化物质的损失程度。同时研究了桃金娘中多酚氧化酶（PPO）和过氧化酶（POD）在不同pH和温度下的稳定性。温度达到100℃、pH小于2时PPO和POD活性才被完全抑制。因此不同的预浸泡方式影响桃金娘果汁PPO和POD的酶活,而抗氧化物质和抗氧化活性的稳定性与PPO和POD的活性负相关。      

67.8ku丝瓜多酚氧化酶酶学性质的研究

目的:为对67.8ku丝瓜多酚氧化酶（PPO）的酶学性质进行研究。方法:从丝瓜中提取、纯化得67.8ku的丝瓜多酚氧化酶,并分别从温度、pH、作用底物、金属离子、抑制剂及表面活性剂对丝瓜PPO活性的影响等方面对丝瓜PPO的酶学性质进行研究。结果:结果表明,pH为6.0时相对酶活最高,pH3.0⁸.0酶活较稳定;其最适作用温度为30℃,对热稳定性较强,70℃保温120min后还具有40%的相对酶活;Co2+、K+、Ca2+等对酶有较强的激活作用,而Na+、Mg2+、Cu2+对酶有明显的抑制作用;抗坏血酸为该酶最好的抑制剂,其次为半胱氨酸、β-巯基乙醇、Na2S2O3、Na2SO3等;十二烷基肌氨酸钠（Laurouyl Sarcosine）、十二烷基硫酸钠（SDS）等对酶有一定的激活作用,而十六烷基三甲基溴化铵（CTAB）、吐温-20（Tween-20）对酶有抑制作用;与l-多巴（l-dopa）的结合力最强,以其为底物时该酶的Km为1.32mmol/L,Vmax为0.22OD475/min。结论:该研究确定了丝瓜多酚氧化酶的最适作用条件及其影响因素,可为控制丝瓜在贮藏、加工过程中的酶促褐变提供理论依据。