Inactivation and conformational change of horseradish peroxidase induced by pulsed electric field

Effects of pulsed electric field (PEF) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. The results showed that activity of HRP decreased with the increase in applied electric field strength (5–25 kV/cm) and pulse numbers (207–1242 pulses). 16.7% and 34.7% reduction of HRP relative activity was achieved immediately after PEF treatment at 25 kV/cm for 207 pulses and 22 kV/cm for 1214 pulses, respectively. The temperature of buffer solution did not increase above 40 °C during PEF treatment. The relative activity exhibited a slow reduction after 24 and 48 h of storage at 4 °C. The HRP conformation changed after PEF treatment, as suggested by CD analysis and fluorescence spectroscopy analysis. α-Helix relative content in the HRP decreased by 35.1% and 57.7% after PEF and heating at 100 °C for 5 min, respectively. The intrinsic relative fluorescence intensity (RFI) increased after PEF treatment. The inactivation of PEF-treated HRP was related to the conformational change of α-helix and the increase of RFI.     

辣根过氧化物酶基因真核表达载体的构建

从辣根侧根中提取总RNA,以OligodT(18)为引物,通过反转录获得了辣根总mRNA的cDNA。以获得的cDNA为模板,利用设计的一对特异寡核苷酸引物P1和P2进行PCR扩增,得到了辣根过氧化物酶(Horseradish Peroxidase,HRP,EC1.11.1.7)同功酶C2的结构基因(hrpC2)。将hrpC2与真核表达质粒载体pPICZα-A分别用限制性内切酶EcoRI和Xbal双酶切后连接,构建了重组质粒表达载体pPICZα-A-hrpC2。将pPICZα-A-hrpC2转化大肠杆菌筛选阳性克隆并测序。测序结果显示,pPICZα-A-hrpC2重组质粒构建成功。利用BlastN程序进行DNA序列分析,显示该序列为目的ORF框,无移码突变;与Fujiyama.K,Takemura.H等报道的hrpC2序列[D90115.1]有95%的同源性。     

辣根过氧化物酶在纤维材料生物整理中的应用研究进展

针对化学法整理纤维材料存在能耗高、纤维损伤大的缺陷,提出借助酶法在温和条件下对纤维材料进行生物整理加工。介绍了辣根过氧化物酶/双氧水/β-二酮类引发剂乙酰丙酮(HRP/H₂O₂/ACAC)三元催化体系的氧化机制,综述了该体系在淀粉、黄麻和丝蛋白生物改性中的应用,包括:通过酶促反应使丙烯酸甲酯与淀粉接枝共聚,提高淀粉浆料对疏水性纤维的成膜性;通过催化黄麻与丙烯酰胺或甲基丙烯酸六氟丁酯接枝,实现黄麻亲水或疏水化整理;通过酶促反应使丝素与丙烯酸接枝共聚,提升丝素材料的仿生矿化效果;通过催化丝胶与甲基丙烯酸甲酯接枝共聚,改善丝胶基生物材料的成型性。指出HRP在纤维整理及生物材料制备中具有潜在的应用前景。     

Characterisation of an anionic peroxidase from horseradish cv. Balady

An anionic peroxidase POIII, molecular weight 56 kDa, was purified from the roots of horseradish cv. Balady. The enzyme exhibited high activity towards o-phenylenediamine and guaiacol, while o-dianisidine had moderate peroxidase activity. Pyrogallol and p-aminoantipyrine had low affinity toward POIII. POIII was found to have a temperature optimum at 40 °C; the enzyme activity remained stable up to 40 °C and retained 87%, 51% and 29% of its activity at 50, 60 and 70 °C, respectively. The enzyme exhibited more than 50% of activity in the pH range between 4.0 and 8.0 with its pH optimum at 5.5. Several metal cations had partial inhibitory effects toward POIII. Fe³⁺ enhanced the activity of the enzyme by 160% at 5 mM. All the metal chelators caused partial inhibitory effects toward POIII, except for EDTA at 1 mM, which had no effect on the enzyme.     

Oxidative degradation of glyphosate and aminomethylphosphonate by manganese oxide

Glyphosate (N-(phosphonomethyl)glycine), the most commonly used herbicide worldwide, degrades relatively rapidly in soils under most conditions, presumably by microbial processes. The most frequently detected degradation product in soil and water is AMPA (aminomethylphosphonic acid). We report the first evidence for an abiotic pathway of glyphosate and AMPA degradation under environmentally realistic conditions. Both glyphosate and AMPA degraded at 20 degrees C in dilute aqueous suspensions of birnessite, a manganese oxide common in soils, as evidenced by the accumulation of orthophosphate in solution over a period of several days. It is concluded that the abiotic degradation involved C-P bond cleavage at the Mn oxide surface, although evidence for C-N bond cleavage in the case of glyphosate and sarcosine, a likely degradation product of glyphosate, was found. The degradation of glyphosate was faster than that of AMPA, and higher temperature (50 degrees C) resulted in faster degradation of both glyphosate and AMPA. The addition of sulfate to the solution had no marked effect on the reaction rate, although Cu2+ addition inhibited degradation. As this metal ion complexes strongly with glyphosate, the inhibition can be attributed to the ability of Cu2+ to limit glyphosate coordination to reactive oxidation sites at the Mn oxide surface. Using a similar experimental design, we were unable to detect glyphosate degradation in an equimolar solution of MnCl2 (0.5 mM). However, we demonstrated that the oxidation of Mn2+ is enhanced both in solution and on an inert surface, in the presence of glyphosate (4:1 Mn-glyphosate molar ratio). This result suggests that the oxidative breakdown of glyphosate in the presence of Mn2+ may ultimately occur following the spontaneous oxygen-mediated oxidation of manganese.     

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.     

The influence of water activity on thermal stability of horseradish peroxidase

The thermal stability of horseradish peroxidase in the solid state was studied as a function of water activity, from 0.11 to 0.88. At all activities the enzyme was found to be much more stable in the solid state than in solution. Inactivation temperatures were in the range of 140–160°C. Inactivation curves show a biphasic behaviour which can be described by a model assuming two fractions (heat labile and heat stable) with independent first order inactivation kinetics. The labile fraction represents approximately 30% of the total activity. The z-value for both stable and labile fractions depends on water activity (moisture content) and has a maximum at a_w= 0.76 (44.4°C and 43.8°C, respectively).     

Purification of a basic peroxidase isoenzyme fromCapsicum fruits and the immunoinhibition of its capsaicin oxidation capacity by antibodies raised against horseradish peroxidase

Pepper fruits contain a peroxidase isoenzyme of basic pI, the peroxidase isoenzyme B₆, located in vacuoles and the principal component of peroxidase polymorphism in the whole fruit. This isoenzyme was purified by preparative isoelectric focusing in glycerol-stabilized 3.0–10.0 pH gradients and characterized for its ability to oxidize capsaicin (8-methyl-N-vanillyl-6-nonenamide). Spectrophotometric studies illustrated that the capsaicin oxidation by pepper peroxidase isoenzyme B₆ was H₂O₂-dependent and was totally abolished by antibodies raised against horseradish peroxidase. From these studies, it can be concluded that capsaicin is oxidized by pepper peroxidase isoenzyme B₆, thus confirming a role for this peroxidase isoenzyme in capsaicin turnover and degradation.     

Inactivation and reactivation of horseradish peroxidase treated with supercritical carbon dioxide

Effects of supercritical carbon dioxide (SCCO₂) on the activity of horseradish peroxidase (HRP) in pH 5.6 acetate buffer solution were investigated. SCCO₂ treatment could effectively inactivate HRP. Higher pressure, higher temperature, and longer treatment time caused more inactivation. The maximum reduction of HRP activity reached nearly 90% at 30 MPa and 55 °C for 60 min. Analysis of first-order reaction kinetic data (characterized by a rate constant k and by a decimal reduction time D) showed that D value was closely related to the pressure and temperature of SCCO₂ treatment. Higher pressures or higher temperatures resulted in lower D values (higher k), the D value of HRP was minimized to 64.52 min treated by the combination of 30 MPa and 55 °C. The Z _p, representing the range of applied pressure between which the D values change by a factor of 10, was 114.81 MPa. The activity of HRP treated by SCCO₂ was reactivated significantly after initial 7-day storage at 4 °C apart from the samples at 30 MPa for 60 min, indicating the HRP inactivation may be reversible and the reactivation of HRP is dependent on the pressure level and treatment time.     

仿生硅化固定化葡萄糖氧化酶与辣根过氧化酶研究

采用分步法将仿生硅化与脂质体技术相结合,模拟细胞微结构,以卵磷脂为原料,制备脂质体微囊,以胺类为诱导剂,在其表面硅化形成氧化硅壳层,实现对葡萄糖氧化酶（GOx）与辣根过氧化酶（HRP）双酶固定。研究了超声时间、诱导剂PDADMA用量、硅前驱体水解液（TMOS）用量、Triton X-100浓度等因素对固定化酶活性影响。结果表明,在200μL PDADMA、0.7 m L TMOS、超声50 min制得的固定化酶活性最高,能迅速硅化形成直径200 nm左右的球形微囊。在此优化条件下制备的固定化酶经重复使用7次,依然达到初始催化酶活的61.46%,经一个月冷藏后酶活仍能达到最初的74.1%。可见,经过固定化,双酶系统的稳定性得到了显著提高。      

辣根过氧化物印迹酶的活性和动力学研究

对辣根过氧化物酶(HRP)进行了生物印迹,并对影响印迹酶活性的因素和印迹后酶的动力学进行了研究。结果表明,印迹酶比游离酶催化活性提高了约6倍,催化效率增加约10倍,Km值减小约3倍。     

聚中性红辣根过氧化物酶传感器测定啤酒中的过氧化氢

制备了以电子媒介体聚中性红固定辣根过氧化物酶(HRP)的生物传感器,采用循环伏安法对该传感器的性能进行了研究。结果表明,辣根过氧化物酶在该电极上实现了稳定的直接电子转移反应,传感器在H2O2浓度为3.18×10-8～3.18×10-3mol/L的范围内具有良好的线性相关性,检出限为6.36×10-9mol/L。应用于啤酒中H2O2的测定,回收率为88.5%～98.6%。     

Properties of bovine gelatin as affected by a cross-linking induced by horseradish peroxidase,glucose oxidase and glucose

Gelatin is an important protein-based hydrocolloid, providing excellent gelling properties in processed foods. Horseradish peroxidase (100 U/g protein), glucose oxidase (1 U/g protein), and glucose (0.025 mmol/g protein) were applied to cross-link bovine gelatin at 37 °C for 3 h, and the gelling properties of cross-linked gelatin were evaluated. Obviously, the cross-linking process did not change the composition of amino acids. The cross-linked gelatin had 39.2% increase in surface hydrophobicity, 54.7% increase in emulsion stability index, but 28.2% decrease in emulsifying activity index, in comparison with bovine gelatin. Laser scanning confocal microscopy observation indicated that cross-linked gelatin rather than bovine gelatin conferred better droplet stability on the generated emulsion after long-storage period of 48 h or 7 days. Moreover, cross-linked gelatin had decreased in vitro digestibility during pepsin hydrolysis (38.5%) and pepsin–trypsin hydrolysis (14.5%) than bovine gelatin. Finally, the gelling and melting temperatures of cross-linked gelatin were 2 °C higher than those of bovine gelatin. Overall, the cross-linking process of bovine gelatin led to easier gelation and improved heat resistance of the subsequently gels.     

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 μM isopropyl-β- -thiogalactopyranoside (IPTG) at 37°C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.     

Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide

The influence of supercritical carbon dioxide (SCCO₂) at 55 °C on inactivation of horseradish peroxidase (HRP) in buffer solution, pH 5.6, was studied while its structural change was analyzed by far UV-circular dichroism (CD) and tryptophan fluorescence spectroscopy. SCCO₂ treatment had significant effects on the residual activity of HRP, the least residual activity was only 12% at 30 MPa. HRP’s secondary and tertiary structures were changed. The α-helix relative content in the secondary structure decreased and the intrinsic relative fluorescence intensity (RFI) increased as the pressure of SCCO₂ treatment was elevated. The HRP’s inactivation closely corresponded to the loss of α-helix relative content and the increase of RFI. After a 7-day storage at 4 °C, the restoration of residual activity and the reversion of the α-helix relative content were observed while RFI resumed with exception of the 30 MPa treatment.     

Steady-state oxidation model by horseradish peroxidase for the estimation of the non-inactivation zone in the enzymatic removal of pentachlorophenol

A theoretical model for the rate of oxidation of pentachlorophenol (PCP) catalyzed by horseradish peroxidase (HRP), was investigated to account for the influence of hydrogen peroxide (H2O2) concentration on the catalytic activity. To evaluate the maximum allowable H2O2 concentration, a relatively simple steady-state model was developed based on the Ping-Pong Bi-Bi mechanism considering the effect of excess H2O2. Several sets of experimental data obtained from batch reactions using an equimolar concentration of H2O2 and PCP were used to estimate the kinetic parameters by a nonlinear regression method. The model profiles acquired using the estimated parameters were in good agreement with experimental data at different initial enzyme and substrate concentrations. The best-fitted parameters were used to predict the initial rate of the enzyme reaction. The model prediction was coincident with the experimental results of other studies, indicating that the proposed model could be used for the optimization of reaction conditions. The maximum allowable H2O2 concentration to prevent H2O2 inhibition was calculated from the proposed model equation: [H2O2](0,max) = (square root)KmH2O2Ki[PCP]0/KmPCP+[PCP]0. Using this equation, a curve depicting the non-inactivation zone for the two substrates (hydrogen peroxide and PCP) was plotted and it could be used for experimental design and optimal process operation. To minimize enzyme inactivation by H2O2, it was determined that the concentration of H2O2 should be lower than 2.78 mM, regardless of the stoichiometric ratio.     

Oxidative and non-oxidative degradation pathways of L-ascorbic acid

L-Ascorbic acid (AA) is an essential component for organisms and is responsible for various functions in the human body. Its structure is in a way that suffering significant structural changes during storage. Herein, the stability of L-ascorbic acid aqueous solution at various thermal (25 and 90 °C ± 2) and atmospheric oxygen or argon was monitored by optical rotation changes as a function of time intervals. Under oxygen, specific optical rotation [α] of the solution was decreased from + 22.0 to + 2.0 after 32 days and under argon + 11.5 after 9 months at 25 °C. The loss of [α] increased at 90 °C under both oxygen and argon atmospheres. Subsequently, the obtained reaction mixtures were evaporated to dryness and analysed by FT-IR, ¹H-NMR and GC-MS techniques. Results of these analyses indicate that AA was decomposed to a complex mixture of reaction products.     

Inactivation kinetics of horseradish peroxidase in organic solvents of different hydrophobicity at different water contents

The thermal stability of horseradish peroxidase suspensions was studied in three organic solvents of different hydrophobicity (dodecane, octane, and 1-octanol) at three different water contents (14.1, 55.3 and 256.2mg water g⁻¹ dry protein). In these conditions, the enzyme is much more stable than in aqueous solutions (inactivation temperatures were in the range of 125–150°C). The enzyme showed a similar stability when in the presence of organic solvents, compared to the enzyme in a solid matrix without organic solvents with the same water content. The inactivation kinetics was well described by assuming the existence of two iso-enzymes, both inactivating according to a First order model. The lowest value for the z-value of both fractions (around 15°C) was obtained at the higher water content studied. The use of solvent and water content variables should be adequate to develop time-temperature integrators to monitor thermal processes at 100–140°C.