重组猕猴桃果胶甲酯酶抑制剂发酵条件优化

在获得含高拷贝猕猴桃果胶甲酯酶抑制剂基因的重组菌株GS115基础上,通过分析甘油加入量、甲醇添加量、山梨醇与甲醇添加比例、诱导初始p H、诱导时间、培养基类型对重组猕猴桃果胶甲酯酶抑制剂(kw PMEI1)表达量的影响,优化了摇瓶发酵条件。结果表明:甘油加入量3%(v/v)、甲醇添加量1%(v/v)、山梨醇溶液(100%,w/v)和甲醇(分析纯)以体积比为1∶1的比例添加、p H5.5、在BMMY培养基中诱导表达5 d为最佳条件。该重组蛋白kw PMEI1的表达量最高可达700.302 mg/L。      

超高压与重组果胶甲酯酶抑制剂联合应用对鲜榨橙汁果胶甲酯酶活性及品质的影响

为了探究超高压与重组果胶甲酯酶抑制剂(recombinant pectin methylesterase inhibitor,rPMEI)联合处理对鲜榨橙汁中果胶甲酯酶(pectin methylesterase,PME)活性及品质的影响,研究了超高压(400、500和600 MPa,5 min,20 ℃)与重组果胶甲酯酶抑制剂对橙汁微生物、PME酶活、色泽和V_C含量的影响。结果表明:超高压处理条件为500 MPa/5 min,rPMEI添加浓度为0.06 mg/mL时,橙汁中的菌落总数、霉菌与酵母菌数均能达到农业行业标准《NY/T 434-2016绿色食品、果蔬汁饮料》所规定的要求,同时PME被完全钝化;橙汁色泽变化显著小于热处理组(ΔE^*=1.22<2.26);V_C保留率为85.1%,显著高于热处理组(保留率=8.33%)。     

果胶甲酯酶抑制剂的研究进展

果胶甲酯酶是植物细胞壁代谢过程中一个重要的酶,参与植物的许多生理过程.果胶甲酯酶的活性调控是非常复杂的过程,在猕猴桃果实中发现了一个对果胶甲酯酶有抑制作用的蛋白,命名为果胶甲酯酶抑制剂,揭示果胶甲酯酶活性调控还存在其他机理.果胶甲酯酶抑制剂在果蔬汁加工业中具有广阔应用前景.综述果胶甲酯酶抑制剂的研究进展,同时对果胶甲酯酶做简要介绍.     

不同发酵条件对重组猕猴桃果胶甲酯酶抑制剂表达量的影响

将已在毕赤酵母中成功表达的重组猕猴桃果胶甲酯酶抑制剂为研究对象,通过改变发酵培养时间、培养基装填量及培养基配方,对该重组蛋白的表达量进行研究。最终确定使用BMMY培养基、表达时间为96h、培养基装填量为10%时为最佳表达条件,在最佳表达条件下,该重组蛋白的表达量最高可达0.2g/L。     

果胶甲酯化修饰与果实质地变化研究进展

甲酯化修饰发生在果胶主链的自由羧基,与果实质地变化密切相关。近年来,基于模式植物的研究揭示:果胶甲酯化修饰在植物生长发育和抗逆等生物学途径中具有关键调控作用,然而,果胶甲酯化修饰在果实领域的研究尚处于起步阶段。本文从果胶甲酯化修饰的概况、研究方法、生物学功能,与果实质地变化的关系及调控机制5个方面综述果胶甲酯化修饰与果实质地变化的研究进展,旨在为相关研究提供参考。     

用GAP启动子在毕赤酵母中组成型表达重组 猕猴桃果胶甲酯酶抑制剂

目的构建组成型表达重组猕猴桃果胶甲酯酶抑制剂(kiwi pectin methylesterase inhibitor, kwPMEI)的毕赤酵母(P．pastoris)GS115工程菌株,探索碳源(葡萄糖、甘油、甲醇)对重组菌表达kwPMEI的影响,纯化kwPMEI并鉴定其对番茄果胶酶的抑制活性。方法应用PCR方法从P．pastoris GS115染色体中扩增了三磷酸甘油醛脱氢酶启动子(pGAP),以其取代诱导型表达载体pPIC9K-kwPMEI上的醇氧化酶启动子(pAOX1),构建了组成型表达载体pGAP9K-kwPMEI,并转化至GS115中。用Tricine-SDS-PAGE和Western blot分析目的蛋白表达情况,镍柱亲和层析纯化目的蛋白,并用凝胶扩散方法鉴定其抑制活性。结果重组毕赤酵母工程菌株成功组成型表达了kwPMEI,48h即达到最大表达水平,表达量约为66 mg/L。并且以甘油为碳源时kwPMEI表达量最高。成功分离纯化了kwPMEI,并经凝胶扩散方法检测表明其具有抑制活性。结论成功构建了组成型分泌表达kwPMEI的毕赤酵母菌株,为kwPMEI在果蔬汁中的进一步应用奠定了基础。     

果胶甲酯酶抑制剂的异源表达及其在果酒降甲醇中的应用

该文从猕猴桃中克隆出果胶甲酯酶抑制剂（Pectin Methylesterase Inhibitor,PMEI）基因,经EcoRⅠ/XbaI双酶切后连接到表达载体pPICzαA上,电转化到毕赤酵母GS115筛选出阳性菌株GS115/pPICzαA-PMEI,并将重组酵母表达的PMEI浓缩纯化后应用于果酒发酵。研究结果表明：重组毕赤酵母表达菌株GS115/pPICzαA-PMEI成功构建,PMEI的发酵表达量为35.38 mg/L。将PMEI应用到果酒发酵中,桔子酒甲醇降低47.54%,含量由189.75 mg/L降低到99.55 mg/L;苹果酒甲醇降低21.52%,含量由118.15 mg/L降低到91.20 mg/L;葡萄酒甲醇变化不显著,含量均低于20.00 mg/L,表明PMEI对果胶丰富且内源性果胶酶强的桔子具有明显的降甲醇效果。桔子酒发酵工艺优化的PMEI最低抑制质量浓度8.84 mg/L,最适温度18 ℃,此时桔子酒中的甲醇降低66.40%到89.10 mg/L。这为低甲醇果酒的发酵提供了新的技术路线。     

果胶甲酯酶对保持苹果质构作用的研究

从质构、酯化度、水溶性果胶含量三个方面研究了果胶甲酯酶对苹果块质构的保持作用。实验证明,用果胶甲酯酶能有效地保持苹果的质构。由经果胶甲酯酶处理的苹果切块（酶浓度0.4mL／50g水,Ca2+浓度0.2％,pH4.5,在0.09MPa下脱气20s）制成的悬浮饮料,在37℃、RH为50％的环境下放置30d后,其硬度保持率为48.5％,咀嚼度保持率为33.46％;未经该酶处理的样品的相应指标分别为32.5％和16.45％。同时模拟实验也表明,苹果中果胶的酯化度从78.6％下降到48.7％,果胶物质在介质中的溶出率从38.95％下降到15.59％。      

果胶甲酯酶对保持苹果质构作用的研究

从质构、酯化度、水溶性果胶含量三个方面研究了果胶甲酯酶对苹果块质构的保持作用。实验证明,用果胶甲酯酶能有效地保持苹果的质构。由经果胶甲酯酶处理的苹果切块(酶浓度0.4mL／50g水,Ca2 浓度0.2％,pH4.5,在0.09MPa下脱气20s)制成的悬浮饮料,在37℃、RH为50％的环境下放置30d后,其硬度保持率为48.5％,咀嚼度保持率为33.46％;未经该酶处理的样品的相应指标分别为32.5％和16.45％。同时模拟实验也表明,苹果中果胶的酯化度从78.6％下降到48.7％,果胶物质在介质中的溶出率从38.95％下降到15.59％。     

绿茶制造中果胶甲酯酶活性的变化

本文探讨了存在于茶树新梢中的果胶甲酯酶(PE,E.C.3.1.1.11)对绿茶品质形成的影响。结果表明:PE影响绿茶中水溶性果胶的含量,对果胶水解产物的形成有着决定性的作用,PE活性与新梢嫩度、品种有关,并随新梢伸育程度的提高而增大,继而又因新梢的进一步成熟而降低;在绿茶制造过程中,PE活性受摊青时间、杀青方式、杀青温度及杀青时间的影响。     

Carrot pectin methylesterase and its inhibitor from kiwi fruit: Study of activity, stability and inhibition

Carrot pectin methylesterase (PME) and its inhibitor (PMEI) from kiwi fruit were successfully purified by affinity chromatography. Enzyme and inhibitor activity and stability and PME–PMEI complex formation, as influenced by intrinsic product factors (pH and NaCl) and extrinsic process factors (temperature and pressure), were studied. The effect of temperature- or pressure-induced denaturation of PME and PMEI on their respective activities was assessed by estimating inactivation kinetic parameters. PME inactivation obeyed first-order kinetics. The enzyme was rather heat-labile but pressure-stable. PMEI inactivation was best described by a model taking into account a processing-stable PMEI intermediate. The behavior of PME and the PME–PMEI complex at elevated temperature or pressure in the presence of pectin was explored by following methanol formation as a function of treatment time. PME catalytic activity was stimulated up to a certain temperature or pressure level before declining. No conclusive evidence was obtained for a temperature-induced dissociation of the PME–PMEI complex, whereas high pressure exposure caused the complex to separate.

Industrial relevance

PME activity control is a major point of interest in the quest of obtaining high quality plant-derived food products. The current study demonstrates that both traditional thermal processing and novel high hydrostatic pressure processing allow stimulation as well as inactivation of PME and, hence, directing the PME-catalyzed pectin hydrolysis. An alternative or additional approach to control endogenous PME activity (e.g. to obtain cloud-stable juices) is through enzyme inhibition using kiwi PMEI. In this context, pH and NaCl boundaries for application were established, the existence of a temperature- and pressure-stable PMEI intermediate was shown and the PME–PMEI complex was proven not to be dissociated at mild temperature and pressure levels. These observations endorse the possibility of inhibiting undesirable PME activity remaining after mild processing.     

Plant pectin methylesterase and its inhibitor from kiwi fruit: Interaction analysis by surface plasmon resonance

Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ? 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.     

Inhibition of tomato pectin methylesterase by partially purified kiwi pectin methylesterase inhibitor protein

The inhibition of tomato pectin methylesterase (PME) by a recently discovered kiwi pectin methylesterase inhibitor (PMEI) is described. PME was consequently purified by CM Sephadex C-50, Concanavalin A-Sepharose 4 B and Mono S chromatography, and PMEI by Q-Sepharose and Sephacryl S-200 chromatography. Inhibition of tomato PME activity under optimal conditions (0.125 m NaCl, pH 7.5) by partially purified kiwi PMEI (MW of 27 kD, pI ≥ 3.67) was independent of the PMEI/PME ratio between 36 and 61% of the maximal uninhibited activity. the non-competitive inhibition observed was optimal in the pH range of 5 to 7. PMEI was inactivated by heating to 120°C, and showed actinidin-like activity towards N-α-benzyloxycarbonyl-L-lysine p -nitrophenyl ester (CBZ-lys-ONp) and azocasein which was partially inhibited by the protease inhibitor leupeptin.     

果胶甲酯酶对番茄丁品质的改善作用分析

选用番茄为原料,探讨了果胶甲酯酶和外源钙离子对番茄丁硬度的影响,并在此条件下考察了果胶甲酯酶、氯化钙、乳酸钙分别单独处理以及果胶甲酯酶分别结合氯化钙、乳酸钙处理对番茄丁弹性、硬度、胶着性和咀嚼度的影响。结果表明:果胶甲酯酶结合氯化钙处理对番茄丁硬化效果最为显著,在添加0.3‰果胶甲酯酶、2‰氯化钙(以番茄丁质量计)时,番茄丁硬化效果最优。相较于对照组番茄丁硬度提高了2倍;其咀嚼度和胶着性也得到了明显的改善。      

Texture improvement of fresh and frozen mangoes with pectin methylesterase and calcium infusion

BACKGROUND: A major problem of mango products is texture loss. The effect of commercial pectin methylesterase (PME) and calcium infusion on improvement of the texture of both fresh and frozen‐thawed mango cubes was investigated in the present study. RESULTS: The weight gain and moisture content of mango samples were greater at relatively high vacuum level (10 kPa). The PME activity of samples infused with PME and calcium at 10 kPa increased fourfold in comparison with that of control and water‐infused samples. The combined effect of PME and calcium was found to improve the texture and microstructure of both fresh and frozen‐thawed mangoes. Fresh mangoes infused with PME and calcium at 10 kPa showed significantly higher firmness than control fresh samples. Frozen‐thawed mangoes infused with PME and calcium at 50 kPa and atmospheric pressure had superior texture and microstructure in comparison with control frozen‐thawed samples. CONCLUSION: The results of the present analysis allow for a better appreciation of the role of PME, calcium and appropriate infusion conditions in improving the texture of both fresh and frozen‐thawed mangoes. Copyright © 2012 Society of Chemical Industry     

Texture improvement of fermented minced pepper under vacuum impregnation with pectin methylesterase and CaCl2 during fermentation

Fermented minced pepper (FMP) usually suffers from the deterioration of texture quality during fermentation, which can affect sensory and consumer acceptance. In this study, vacuum impregnation (VI) with CaCl₂, pectin methylesterase (PME) and CaCl₂ and PME (PME + CaCl₂ + VI) were compared to improve the texture quality of FMP. FMP treated with PME + CaCl₂ + VI showed the relatively intact cells structure after fermentation. In that case, its firmness maintained high level, while water-soluble pectin (WSP) content was minimum after fermentation. Meanwhile, the molar ratio of most monosaccharides of WSP in PME + CaCl₂ + VI treated FMP decreased, while rhamnose (Rha) molar ratio significantly (p < 0.05) increased after fermentation. The high Rha content represents the stability of rhamnogalacturonan-I linear skeleton of WSP. The negative effect on molecular weight of WSP was delayed by PME + CaCl₂ + VI treatment, and its peak area and value increased after fermentation. Atomic force microscope images indicated that PME + CaCl₂ + VI treatment could retain the long chain and branch structures, and inhibit the degradation of WSP net-like structure at some extent. Hence, PME + CaCl₂ + VI treatment was effective to improve the texture of FMP and inhibit the solubilisation of WSP via the formation of cross-linked pectin chains between Ca²⁺ and demethylesterified pectin.     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry