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.     

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。     

促进植物内源性果胶甲酯酶催化作用的因素与机制及其在果蔬加工中的应用

植物内源性果胶甲酯酶（pectin methylesterase,PME）存在于天然植物组织中,可以催化高酯果胶脱甲基酯化生成低酯果胶,进而影响果蔬硬度、出汁率等质构及加工品质。一方面,为了避免贮藏期间出现果蔬汁浑浊沉淀、果蔬罐头中果肉变软、调味酱（如辣椒酱）中皮肉分离等不良品质,会在加工过程中抑制PME活性;另一方面,为了提高果蔬汁出汁率、生产低酯果胶等,也可在食品中添加外源PME。目前,对于通过激活内源PME来改善食品加工的研究较少,因此,本综述重点讨论促进植物内源性PME催化作用的因素与机制及其在果蔬贮藏加工中的应用,为植物内源性PME在果蔬加工中的充分利用提供理论参考。     

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.     

气调贮藏对‘红阳’猕猴桃果胶含量及相关酶活的影响

研究了气调对采后‘红阳’猕猴桃贮藏期间果实硬度、果胶(可溶性果胶、原果胶)含量及果胶代谢相关酶(果胶甲酯酶PE、多聚半乳糖醛酸酶PG和纤维素酶CX)活性的影响。在温度(1±0.5)℃、相对湿度80%⁹8%的条件下,以空气为对照(CK),采用了4种不同的气调组分CA1(2%O2+3%CO2)、CA2(2%O2+6%CO2)、CA3(5%O2+3%CO2)、CA4(5%O2+6%CO2)。试验表明:红阳猕猴桃后期贮藏过程中,果实软化与PG和CX有密切联系,而与PE可能无直接联系。气调贮藏可显著延长果实贮藏期,其中CA1和CA3处理的果实贮藏期长达120 d,而CA1可更有效地延缓果实硬度的下降,抑制细胞壁降解酶的活性,更好地保持‘红阳’猕猴桃的商品性。     

果胶甲酯酶的作用机理及在食品加工中的应用

该文综述食品加工中应用较为广泛的果胶酶的一个分类——果胶甲酯酶的作用机理及目前在果蔬汁澄清、果蔬制品改善、低酯果胶制备等食品相关行业中的应用现状,并进行总结,为果胶甲酯酶在食品加工中的深入研究开发及未来应用前景预测提供参考。     

高产高纯度果胶甲酯酶黑曲霉工程菌的构建

为获得一株高产高纯度的果胶甲酯酶的黑曲霉工程菌,提高果胶甲酯酶的产量,从果胶酶生产菌种中克隆了果胶甲酯酶基因pmeA,通过同源重组的原理,冻融法转化农杆菌、农杆菌介导法转化黑曲霉方法,成功构建了分泌表达果胶甲酯酶的纯合重组菌株TH-2（glaA::pmeA）。基本发酵培养基中发酵第9 d上清中最高酶活达到467.77 U/mL。进一步敲除重组菌株TH-2（glaA::pmeA）中背景蛋白酸稳定的α-淀粉酶的编码基因asaA,获得纯合重组菌株TH-2（glaA::pmeA?pyrG?asaA）。该菌株在添加1%的硫酸铵的发酵培养基中培养7 d后,发酵液上清中主要的背景蛋白均消失。但是与纯合重组菌株TH-2（glaA::pmeA）相比,果胶甲酯酶表达量有所下降,最高酶活为255.40 U/mL。重组果胶甲酯酶的最适作用温度为50 ℃,适合的温度范围是40~80 ℃,在80 ℃下仍能维持其酶活性的70%以上,适合的pH范围是3.0~5.0,最适pH为4.0。最终获得了一株温度和pH作用范围较宽的高产高纯度果胶甲酯酶的黑曲霉工程菌。     

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。     

Demethylation of Pectic Substances: Relationship to Methylesterase Activity during Brine Storage of Cherries

Residual pectin methylesterase activity (60% of initial activity) was present in cherry fruit stored 12 mo in brine. The specific activity of pectin methylesterase increased during the first 4 mo (195% of initial specific activity) but remained relatively stable thereafter. Methanol concentration increased (+144.5%) indicating that pectic substances underwent continuous demethylation during 12 mo storage. Pectin deesterification was probably of enzymatic origin since notable chemical deesterification occurred only at a pH lower than that found in fruit during storage. Residual pectin methylesterase activity introduces the possibility of improving fruit texture by manipulating physico-chemical conditions prior to blanching and candying.     

ACEROLA's CLONES OF INDUSTRIAL INTEREST

In order to know which clone of acerola is better for acerola industrialization, we studied the pectin methylesterase (PME) specific activity, pectin content and vitamin C content in five different clones of acerola. The pectin yield varied from 1.37 to 2.99% and the highest content of pectin occurred in clones 3 and 5. Ascorbic acid varied significantly from 1157.5 to 1735.5 mg/100 g of pulp in the five clones. The highest content of vitamin C occurred in clone 4. The PME specific activity varied from 0.79 to 2.92 units g ^?1 /g of pulp and the highest values occurred in clone 2. We also studied the optimum temperature and the optimum pH of this enzyme. Clones 1, 2, 4 and 5 showed optimum temperature at 90C. Clone 3 showed practically the same specific activity at all temperatures studied. Clones 1 and 4 showed an optimum pH of 9.0 and clone numbers 2, 3 and 5 showed a pH optimum at 8.5.     

低盐腌渍仔姜果胶甲酯酶保脆工艺的优化

采用果胶甲酯酶对低盐腌渍的仔姜进行保脆处理。采用单因素试验研究葡萄糖酸钙的添加量、果胶甲酯酶的添加量和保脆处理的温度与时间对低盐腌渍仔姜的影响。以硬度为指标,通过正交试验优化低盐腌渍仔姜的保脆工艺。结果表明,低盐腌渍生姜的最佳保脆工艺条件为果胶甲酯酶添加量0.8%,处理温度40℃,处理时间25min,葡萄糖酸钙添加量0.4%。在此条件下,保脆后的低盐腌渍仔姜片的平均硬度为3209.224g。     

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.     

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6–9) and moderate thermostability (optimum ca. 70 °C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.     

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.