基于环氧功能基团的杂化硅胶固定化酶反应器

在蛋白质组学研究中, 为解决自由溶液酶解时间长、蛋白酶自降解等问题, 各种类型的固定化酶反应器引起了人们的关注. 采用溶胶凝胶法, 在100 µm内径毛细管内, 以3-(甲基丙烯酰氧)丙基三甲氧基硅烷为功能单体, 以四乙氧基硅烷为交联剂, 制备了一种新型的杂化硅胶整体材料. 并通过在整体柱表面由环氧功能团水解得到的二醇与胰蛋白酶(trypsin)的氨基进行一步反应, 实现了胰蛋白酶固定化. 利用该IMER, 在47 s内实现了牛血清白蛋白的酶解; 经反相色谱分离和质谱鉴定, 序列覆盖率在35%以上, 与自由溶液酶解12 h的效果相当. 结果表明, 基于亲水性杂化硅胶整体材料的IMER有望在蛋白质组学研究中发挥重要作用.     

大鼠肝脏过氧化氢酶的分离纯化及性质

根据过氧化氢酶的催化特性,采用盐析,透析,离心等技术,从大鼠肝脏中分离纯化过氧化氢酶,提纯倍数达到26倍,酶活性回收率为57%。为一般实验室分离纯化大鼠过氧化氢酶提供了一种有效的方法。酶学性质研究表明,该酶最适温度为37℃,最适pH值为7.5,在此条件下,以过氧化氢为底物的Km值为56 mmol.L-1。     

ALG-SiO2杂化凝胶固定化多酶体系催化CO2转化甲醇研究

采用甲酸脱氢酶（FateDH）、甲醛脱氢酶（FaldDH）和醇脱氢酶（ADH） 3种脱氢酶为催化剂,以原型烟酰胺腺嘌呤二核甙酸（NADH）作为电子供体,通过3步连串反应将CO2转化为甲醇。采用正硅酸甲酯原位水解方法将二氧化硅掺杂于海藻酸（ALG）溶胶中,通过双交联制备出新型ALG-SiO2杂化凝胶。与ALG凝胶相比,ALG-SiO2结构更加紧密,酶的泄漏率大大降低,酶活性得以很好保持。将酶包埋于ALG-SiO2后,存放60 d以后或重复使用10次以上,酶活性都能保持80%以上,与之相比,包埋于ALG凝胶中的酶活性几乎完全丧失。     

乙基纤维素微胶囊对过氧化氢酶固定化的研究

以乙基纤维素作膜材,用液中干燥法使过氧化氢酶微胶囊化。研究了微胶囊化操作条件对酶活性的影响。通过测定微胶囊化酶的释放曲线,证明微胶囊膜对过氧化氢酶具有较好的固定性能力。固定化酶用于催化底物过氧化氢分解,测定米氏常数为０．５５ｍｏｌ／Ｌ。     

基于金属有机框架的固定化酰胺酶制备及催化合成(S)-4-氟苯甘氨酸

以微水溶剂热法快速制备的稳定锆基金属有机框架为载体,戊二醛为交联剂,采用交联法对酰胺酶进行固定化,考察了不同条件对酰胺酶固定化效率的影响.结果 表明,戊二醛浓度为1.0％、交联时间为180 min、载体与酶的质量比为8∶1,固定化效率最佳,固定化酶活力回收率达86.4％,蛋白负载量达115.3 mg/g.固定化酶最适温...     

Thermus thermophilus HB27锰过氧化氢酶在大肠杆菌中的表达及酶学性质分析

碱性嗜热过氧化氢酶是一种重要的纺织用酶。根据大肠杆菌密码子使用偏爱性,对Thermus thermophilus HB27来源的含锰过氧化氢酶基因进行密码子优化,将优化后的基因连接至表达载体pET28a(+)上,转化到E.coli BL21(DE3)中进行诱导表达。结果表明在含有14mmol/L Mn2+浓度的培养液中以0.2 mmol/L的IPTG 42℃条件下诱导2 h的情况下,菌体破碎上清液中的酶活力可达25 U/ml。利用Ni亲和层析柱对该Mn-CAT进行纯化,酶学性质研究表明:此酶的最适温度为70℃,最适pH为pH 10.0,在80℃保温2 h,酶活力不损失;pH9.0~11.0的环境中放置2 h后,酶活仅损失约10%,此酶具有良好的工业开发潜力。     

谷氨酸棒杆菌过氧化氢酶的异源表达、纯化以及酶学性质

过氧化氢酶能催化过氧化氢分解为水和氧气,在工业上有着较为广泛的应用。然而,纺织和造纸工业的特殊的高碱性环境,使得开发碱性过氧化氢酶有着重要的应用价值。利用大肠杆菌表达来自于谷氨酸棒杆菌的过氧化氢酶,对其表达条件进行了优化,并通过镍柱亲和层析的方法分离纯化重组蛋白,然后表征纯酶的酶学性质。最适表达条件为:诱导剂IPTG浓度0.2 mmol/L,诱导温度25℃,诱导时间11 h。过氧化氢酶比酶活达到55 266 U/mg,具有较高的催化活性。该酶具有相当宽泛的p H值适应范围,在p H 4.0–11.5范围内均具有较高的酶活性,并在p H 11.0条件下表现出最高的酶活性。将纯酶在p H 11.0的溶液中处理3 h时剩余酶活为93%,说明该酶在高碱条件下有良好的稳定性。该酶最适温度为30℃,在25–50℃热稳定性较好。其动力学参数Km为25.89mmol/L,Vmax为185.18mmol/(min?mg)。抑制剂十二烷基硫酸钠(SDS)、尿素、Na N3、β-巯基乙醇、EDTA对酶活有不同程度的抑制作用。来源于谷氨酸棒杆菌的过氧化氢酶具有较高的催化效率、良好的碱耐受性,在工业生产中有较好...     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。     

融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

【目的】利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶Kat A进行改性,以强化重组过氧化氢酶在工业中的应用适应性。【方法】将自组装双亲短肽S1vw通过连接肽PT-linker融合在Kat A的N端,构建重组质粒p HT254-S1vw-PT-kat A,将其与携带天然酶基因的p HT254-kat A分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。【结果】成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-Kat A和天然酶Kat A的最适反应温度均为30°C,最适反应p H值均为11.0。然而,融合酶在p H 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65°C和70°C下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4°C储存14 d后相对酶活为8...     

Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu²⁺) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02 mg mL⁻¹ urease in 10 mM PBS (pH 7.4) at +4 °C) exhibited the highest catalytic activity of ∼2000% and ∼4000% when stored at +4 °C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4 °C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.     

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.     

A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.     

Modulation of the catalytic activity of free and immobilized peroxidase by extremely low frequency electromagnetic fields: dependence on frequency

A study of the influence of electromagnetic fields (EMF) of various frequencies, from 50 up to 400 Hz, on the catalytic activity of soluble and insoluble horseradish peroxidase (POD) was carried out. To simulate the conditions in which the enzyme operates in vivo, the POD was immobilized by entrapment on a gelatin membrane or by covalent attachment on a nylon graft membrane. The rate of inactivation of the soluble POD was found to exhibit positive and negative interactions with the 1 mT applied magnetic field, with an optimum positive effect at 130 Hz. The immobilized PODs, on the contrary, do not exhibit negative interactions, but show a maximum positive interaction at 150 Hz when entrapped and at 170 Hz when covalently attached. At 50 Hz and at frequencies higher than 250 Hz no effects were observed with insoluble POD. The optimum frequency of positive interaction between the EMF and the catalytic activity of the insoluble enzymes is shifted with respect to that of the soluble enzymes towards higher frequencies, the size of the shifts being dependent on the intensity of the physical forces involved in the immobilization process.     

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H₂O₂ to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1?CFU/mL with wide linear ranges (10¹–10⁶?CFU/mL).     

Convenient enzymatic resolution of (R,S)‐2‐methylbutyric acid catalyzed by immobilized lipases

The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)‐2‐methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)‐pentyl 2‐methylbutyrate was prepared in 2 h with c 40%, ee_p 90%, and E = 35, while Thermomyces lanuginosus leads to c 18%, ee_p 91%, and E = 26. The (S)‐enantiomer was obtained by the use of Candida rugosa or Rhizopus oryzae (2‐h reaction, c 34% and 35%, ee_p 75 and 49%, and E = 10 and 4, respectively). Under optimal conditions, the effect of the solvent, the molar ratio, and the nucleophile were evaluated.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

Gene cloning,heterologous expression,in vitro reconstitution and catalytic properties of a versatile peroxidase

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide and amino-acid identity with Pleurotus eryngii gene vpl, which encodes a versatile peroxidase with unique catalytic properties, and only reported in Pleurotus and Bjerkandera species. Re-identification of the supposed L. irina strain revealed that, in fact, it is a P. eryngii strain. The new P. eryngii peroxidase was expressed in Escherichia coli, and the recombinant protein folded in the presence of cofactor to obtain the active form. The purified enzyme was able to oxidize Mn²⁺, veratryl alcohol, substituted phenols, and both low and high redox-potential dyes, demonstrating that it belongs to the versatile peroxidase family (named VPL3). These catalytic properties agreed with the presence of both Mn²⁺ and aromatic-substrate oxidation sites in its molecular structure.