黑曲霉果胶酯酶的研究

黑曲霉(As3.3324)用高甲氧基果胶诱导,当接种量为3×103个孢子,30℃恒温培养,转速90r/min,液体培养4d时果胶酯酶的活力最大,为90.2U/mL培养基。该果胶酯酶能将高甲氧基果胶的酯化度从64.54%降低到45.70%。经果胶酯酶作用后低甲氧基果胶的粘度为0.006Pa·s,与同样条件处理的高甲氧基果胶的粘度0.007Pa·s相差不大。      

黑曲霉果胶酯酶的研究

黑曲霉(As3.3324)用高甲氧基果胶诱导,当接种量为3×103个孢子,30℃恒温培养,转速90r/min,液体培养4d时果胶酯酶的活力最大,为90.2U/mL培养基。该果胶酯酶能将高甲氧基果胶的酯化度从64.54%降低到45.70%。经果胶酯酶作用后低甲氧基果胶的粘度为0.006Pa·s,与同样条件处理的高甲氧基果胶的粘度0.007Pa·s相差不大。     

果胶酯酶制备低酯果胶的研究进展

随着对低糖、低热量食品的需求日益增大,低酯果胶已供不应求。果胶酯酶脱酯制备低酯果胶是一种生产效率极高的方法,对解决国内低酯果胶供不应求的难题将有极大帮助。本文综述了国内外果胶酯酶制备低酯果胶的研究进展。     

果胶酯酶制备低酯果胶的研究进展

随着对低糖、低热量食品的需求日益增大,低酯果胶已供不应求。果胶酯酶脱酯制备低酯果胶是一种生产效率极高的方法,对解决国内低酯果胶供不应求的难题将有极大帮助。本文综述了国内外果胶酯酶制备低酯果胶的研究进展。      

固定化果胶酯酶的研究

采用明胶作载体和戊二醛作交联剂制备固定化果胶酯酶,并对其固定化条件、酶学性质和应用进行了研究。结果发现,明胶浓度为15%、戊二醛为5%、固定化酶量0.15mL(稀释6.7倍),缓冲液pH4.0时制备的固定化酶的酶活和回收率较高,分别是48.725U和68.81%;固定化酶的最适温度45℃,最适pH4.0,Km值0.017%,pH2.8～6.0稳定;果胶酯酶固定化后温度稳定性、贮存稳定性和操作稳定性都明显增强,50℃下保温3h,酶活保留86%;4℃贮存16d,酶活保留90.15%。反复利用6次,酶活仍保存80.99%,半衰期为11.7d。固定化酶作用于高酯果胶不同时间后得到甲氧基含量呈下降趋势的低酯果胶。     

利用果胶酯酶制备低酯果胶工艺研究

研究高酯果胶脱酯工艺的最佳条件;利用果胶酯酶对商品高酯果胶进行脱酯,采用L934正交试验法研究确定酶用量、脱酯温度、pH及脱酯时间对脱酯效果的影响.利用果胶酯酶进行脱酯的最佳工艺参数为:果胶酯酶0.06 g,脱酯温度45℃,脱酯pH 7,脱酯时间70 min,所得果胶甲氧基含量为4.96%.利用酶法制备低酯果工艺简单,脱酯效率较高.     

柑桔汁沉淀与果胶酯酶的关系

现代加工的桔汁都呈混浊状态,混浊物质主要是由不溶性的直径在10微米以下的微细颗粒组成,它们含有大量的色素、风味及营养成分,是柑桔汁色、香、味、体的重要组成部分。但若不进行混浊稳定化处理,混浊物质就会在贮运过程中,以凝絮状态析出,产生清液层,从而失去了柑桔汁应有的鲜艳色泽和混浊状态。这种现象主要是由于果胶酯酶的存在,破坏了胶体的稳定性所引起的。一、果胶酯酶的特性现已发现果胶酯酶有12种单体,因对热的稳定性不同,而分为热敏单体和热稳单体两     

内源果胶酯酶制备低甲氧基果胶的研究

以新鲜橙皮为原料，在盐酸水解乙醇沉淀提取果胶之前，激活并利用果皮中固有的果胶酯酶进行果胶的酶法脱酯，制备低甲氧基果胶，以产品的甲氧基含量和果胶得率为指标，确定最佳工艺条件。结果表明新鲜橙皮内源酶法制备低甲氧基果胶的最佳工艺条件为：加入果皮浆液量0．15％的内源性果胶酯酶激活剂碳酸钠，控制温度45℃，pH值8．0进行脱酯，时间60min；果胶提取温度90％，时间60min，pH值2．0；在此条件下制备的果胶甲氧基含量为5．93％，符合低甲氧基果胶标准，果胶得率为2．46％。     

应用果胶甲基酯酶改善番茄酱的粘度

研究了添加果胶甲基酯酶对不同类型番茄酱粘度的影响。结果表明,果胶甲基酯酶对于热破番茄酱具有明显增强粘度的作用,而对于冷破番茄酱作用较弱。热破番茄酱中的果胶在果胶甲基酯酶的作用下成为低酯果胶,并在钙离子的作用下进一步形成交联,从而使粘度提高;而冷破番茄酱中没有果胶(或含量很低),所以果胶甲基酯酶对其粘度没有明显作用。      

Textural properties of gelling system of low-methoxy pectins produced by demethoxylating reaction of pectin methyl esterase

ABSTRACT:  After deesterification of commercial pectins with a pectin methyl esterase (PME), their gelling properties were characterized using instrumental texture analysis. The final degree of esterification (DE) of the high- and low-methoxy pectins reached approximately 6% after the PME treatment, while deesterification of low-methoxy amidated pectin stopped at 18% DE. Furthermore, DE of high-methoxy pectin was tailored to be 40%, which is equivalent to the DE of commercial low-methoxy pectin. As a result, significant changes in molecular weight (Mw) distribution were observed in the PME-treated pectins. The texture profile analysis showed that PME modification drastically increased hardness, gumminess, and chewiness, while decreasing cohesiveness and adhesiveness of the pectin gels ( P < 0.05). The pectin gel with relatively high peak molecular weight (Mp, 3.5 × 10⁵) and low DE (6), which was produced from high-methoxy pectin, exhibited the greatest hardness, gumminess, chewiness, and resilience. The hardness of low-methoxy amidated pectin increased over 300% after PME deesterification, suggesting that the effects of amide substitution could be reinforced when DE is even lower. The partial least square regression analysis indicated that the Mw and DE of the pectin molecule are the most crucial factors for hardness, chewiness, gumminess, and resilience of gel matrix.     

Pectin methyl esterase treatment on high‐methoxy pectin for making fruit jam with reduced sugar content

BACKGROUND: Pectin methyl esterase (PME) has been postulated to catalyse the transacylation reaction between pectin molecules. The present study aimed to prove the occurrence of this reaction. The feasibility of applying PME‐catalysed transacylation between high‐methoxy pectin molecules in making fruit jam with reduced sugar content was also investigated. RESULTS: PME treatment increased the turbidity and particle size in pectin solution and the molecular weight of pectin, while it decreased the number of methoxy ester linkages and the intensity of the CH₃ absorption peak in the Fourier transform infrared spectrum without changes in the number of total ester linkages in pectin molecules. These findings support the occurrence of PME‐catalysed transacylation between pectin molecules. Higher values of hardness, gumminess and chewiness were found in a jam containing PME‐treated citrus pectin (10 g L^?1) and sugar (350 g L^?1) as compared with either a jam containing untreated citrus pectin (10 g L^?1) and sugar (350 g L^?1) or strawberry jam containing pectin (10 g L^?1) from the fruit and sugar (650 g L^?1). CONCLUSION: The demand for sugar in jam making can be greatly reduced by the use of PME‐treated high‐methoxy pectin. Copyright © 2012 Society of Chemical Industry     

Enhanced inactivation of pectin methyl esterase in orange juice using modified supercritical carbon dioxide treatment

The aim of the study is to quantify the effect of ethanol addition and exposure surface on the inactivation of pectin methyl esterase (PME), a juice clarifying enzyme, in orange juice using supercritical carbon dioxide (SC‐CO₂). Addition of ethanol to the SC‐CO₂ at 2% (v/v) caused greater inactivation than SC‐CO₂ alone, with a maximum reduction of PME activity of 97% at 30 MPa and 40 °C for 60 min. As the surface area to volume ratio was increased, the rate of inactivation of PME increased. Analysis of first‐order reaction kinetic data revealed that D values were greatly influenced by ethanol addition and agitation. With the addition of 2% ethanol, the D value reduced by half, that is, 56 min from 109 min. With impeller agitation of the sample at 1100 ± 100 rpm, the D value for PME was further reduced to 43 and 30 min without and with ethanol, respectively. The activity of PME treated with SC‐CO₂ remained unchanged after 14 days of storage at 4 °C. Treatment did not significantly change pH or colour, but did significantly increase the cloud values of the juice, resulting in a cloud stabilised juice with similar qualities to fresh juice.     

超嗜热细菌Thermotoga maritima酯酶Tm1350克隆、表达及其酶学性质研究

以超嗜热细菌Thermotoga maritima基因组DNA为模板,通过PCR法扩增酯酶Tm1350基因,构建重组质粒p ET15b-Tm1350,在大肠杆菌Escherichia coli BL21（DE3）中实现表达,并采用p-NPA方法测定其酶活性。研究结果表明,酯酶Tm1350可以水解不同碳链长度（10C）的对硝基苯酚酯,其中对硝基苯酚戊酸酯（p NP-C5）是该酶最适合底物;该酶最适反应温度和p H分别为70℃和6.5,90℃处理5h,仍有50%以上的酶活力,具有较强的热稳定性。酯酶Tm1350对金属离子、抑制剂、变性剂和有机溶剂具有较强的抗性。      

甜荞麦16 kDa过敏原基因的克隆及其原核表达载体的构建

目的 对甜荞麦(common buckwheat)过敏原的分子生物学开展研究。方法 通过RT-PCR克隆甜荞麦16 kDa过敏原蛋白的全长基因, 并根据序列设计带有酶切位点的特异性引物, 扩增甜荞麦16 kDa过敏原基因的完整开放阅读框, 与pET-28a载体连接, 构建原核表达载体。 结果 本研究成功克隆了甜荞麦16 kDa过敏原蛋白的基因, 且构建了其原核表达载体。该基因含有长度为450 bp的开放阅读框, 编码149个氨基酸, 在GenBank数据库中的登录号为EU883600, 同源性分析发现其与数据库中已知的荞麦过敏原基因有高度的同源性。结论 本研究为甜荞麦16 kDa过敏原蛋白的重组表达和临床过敏性疾病的诊断奠定了基础。     

Hydrolysis of apple pectin by the coordinated activity of pectic enzymes

The hydrolysis of pectin from apples, cv. Budimka fruit (Pyrus mahus L.), by individual and/or combined action of fungal polygalacturonase from Aspergillus niger (FPG), fungal pectin esterase from A. niger (FPE) and plant tomato pectin esterase (PPE) was studied. The optimum pH values for individual actions of FPG, FPE and PPE on 1% apple pectin were determined to be 4.5, 3.5 and 6.5, and the optimum temperatures were 40, 45, and in the range 45–50 °C, respectively. FPE was found to be more efficient for the hydrolysis of the apple pectin than was PPE from tomato. By measuring the initial velocities on 1% apple pectin it was confirmed that the PG expressed no effect on the PE activity. By using the combination of FPG (162 U/l) and FPE (60 U/l), e.g., in a respective ratio of 2.5, an efficient pectin degradation process, with a viscosity reduction of η/η₀ = 1.05, could be reached in less than 2 h. This process produced about 160 ppm of methanol in the pectin digest. The long term hydrolysis reaction of the apple pectin with FPG (162 U/l) and FPE (27 U/l) achieved a degree of hydrolysis of around 29% after 12 h and consisted mostly of trimers (28.4%).     

枯草芽孢杆菌胺氧化酶基因的克隆与表达

以细菌型豆豉工业发酵菌种枯草芽孢杆菌（Bacillus subtilis）BJ3-2为材料,根据NCBI中菌株B. subtilis 168的胺氧化酶（AMO）基因序列设计同源引物,克隆获得菌株B. subtilis BJ3-2的胺氧化酶基因YobN序列。测序结果显示,YobN基因开放阅读框（ORF）长为1 437 bp,编码478个氨基酸,分子质量为53.79 ku,与菌株B. subtilis 168同源性达98%。目的基因克隆至原核表达载体,获得重组菌pET28a-YobN/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示,浓度为1.0 mmol/L的异丙基硫代半乳糖苷（IPTG）22 ℃诱导5 h,表达量最高达1.30 mg/mL。该研究为AMO活性的研究奠定了基础,对豆类发酵食品中生物胺的控制具有重要意义。     

白酒杂醇油酯化酶的高效表达

该研究提取具有酯化杂醇油能力的近平滑假丝酵母（Candida parapsilosis）的总RNA,逆转录聚合酶链反应（RT-PCR）得到酯化酶的编码序列,构建重组质粒并转化至毕赤酵母（Pichia pastoris）,筛选得到高效表达杂醇油酯化酶的毕赤酵母转化子,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示毕赤酵母转化子表达了分子质量为50 kDa酯化酶。酯化酶在30 ℃酯化底物1 d,乙酸正丙酯含量为14.38 mg/100 mL,乙酸异丁酯含量为5.93 mg/100 mL,乙酸异戊酯含量为2.09 mg/100 mL。高效表达杂醇油酯化酶的毕赤酵母转化子对小曲白酒和液态酿造蒸馏酒的品质提升具有重要的实际意义。