N-糖基化对重组木聚糖酶XynA酶学特性的影响

为实现木聚糖酶的高效表达,扩大实际生产应用,本实验将木聚糖酶基因xynA在毕赤酵母中进行重组表达,同时采用糖基化抑制剂衣霉素处理毕赤酵母细胞,以此来探讨N-糖基化对木聚糖酶XynA酶学性质的影响。结果表明,SDS-PAGE电泳图谱显示木聚糖酶XynA发生了N-糖基化修饰,分子量约为45 kDa,酶活为406.6 U/mL,最适pH及反应温度分别为5.0和65 ℃;而添加不同浓度衣霉素处理后的木聚糖酶最适pH不变,最适反应温度下降5 ℃,随着衣霉素浓度的增加,去糖基化的木聚糖酶酶活力、分泌量及温度稳定性均有所下降,且当衣霉素添加浓度为15 μg/mL时,剩余酶活为53.6%。去糖基化的木聚糖酶耐受胃蛋白酶的能力较糖基化的有所增强,Na+、K+、Li+、Ca2+、Al3+、EDTA相对于糖基化的木聚糖酶表现出了一定的激活作用。以上结果说明N-糖基化作为一种重要的翻译后修饰对木聚糖酶XynA的分泌及热稳定性有促进作用。     

米黑根毛霉脂肪酶基因在毕赤酵母中的高效表达

脂肪酶是主要工业用酶制剂之一,在食品、洗涤剂和制药等领域广泛应用。将编码米黑根毛霉(Rhizo-mucor miehei)脂肪酶RML的基因克隆到pPIC9K载体中,构建了分泌型表达载体pPIC9K-RML,载体经线性化后转化Pichia pastorisGS115,G418梯度筛选获得了分泌表达RML的重组毕赤酵母工程菌,SDS-PAGE分析显示表达的脂肪酶分子量大小与预期一致。初步研究表明,重组脂肪酶最适温度为40℃,最适pH值为8.0,以橄榄油为底物时,发酵上清液酶活最大可达102 U/mL,表明构建的重组毕赤酵母工程菌具有较好的工业化生产潜力。     

康氏木霉纤维素酶CBHI基因克隆及在毕赤酵母中的表达研究

在毕赤酵母中表达康氏木霉(Trichoderma koningii)纤维素酶基因cbhI。提取经诱导的康氏木霉mRNA,通过反转录及PCR反应扩增纤维素酶cbhI基因cDNA。将cbhI基因克隆到毕赤酵母表达载体pPICZαA上,采用电激法将经SacⅠ线性化的重组质粒pPICZαA-cbhI转化毕赤酵母GS115,MD及G418抗性平板筛选cbhI基因高拷贝转化子,并用PCR鉴定转化子。BMMY培养基中加入0.5%甲醇对毕赤酵母进行纤维素酶CBHI诱导表达。SDS-PAGE电泳结果显示,表达的重组蛋白相对分子质量约67 kD,pNPC酶活为24.1 U/L,酶蛋白量为0.22 mg/mL,表明,康氏木霉cbhI可以在毕赤酵母中表达,并具有较高活性。     

α-L-鼠李糖苷酶AnRhaE在毕赤酵母中的表达及应用

α-L-鼠李糖苷酶对天然产物之间的转化具有重要的作用和应用价值.该研究采用毕赤酵母表达 α-L-鼠李糖苷酶AnRhaE,构建pPIC9K-AnRhaE表达载体,电转化至毕赤酵母GS115和KM71H中,经诱导表达,SDS-PAGE电泳显示GS115更有利于生产AnRhaE酶.AnRhaE酶在45～55℃时酶活力高,最适...     

雪白根霉脂肪酶基因在毕赤酵母中的高效表达及其酶学性质研究

采用同源克隆法获得了雪白根霉的脂肪酶基因(rnl),将其连接到pPICZαA载体上,得到重组表达载体pPICZαA-rnl。将表达载体pPICZαA-rnl用限制性内切酶SacI线性化后,电击转入毕赤酵母X-33中。雪白根霉脂肪酶基因在毕赤酵母X-33中成功表达,重组菌株摇瓶培养144 h后,酶活可达48 U/mL。重组酶最适pH值为8.5,最适反应温度为35℃。1 mmol/L的Mn2+,Ca2+和K+以及1%的表面活性剂吐温20,吐温80,曲通100对重组脂肪酶具有激活作用。重组酶的最适底物为三月桂酸甘油酯(C12)。     

外源脂肪酶在毕氏酵母表面展示及发酵过程分析

分别考察基于絮凝素和凝集素2种不同展示系统,展示有外源脂肪酶CALB(Candidan Antarctica lipase B)的重组展示酵母的展示酶活、上清酶活,发现毕赤酵母作为宿主在外源脂肪酶展示过程会分泌目的蛋白至上清中,引起"外泄",进一步通过重组展示酵母外源蛋白鉴定、外源蛋白去糖基化、发酵过程中蛋白变化趋势观察,表明基于絮凝素系统的非共价锚定是引起展示过程中蛋白外泄比例过高的主要原因,研究为实现脂肪酶在毕赤酵母细胞的高效展示进而提高展示酶的催化效率提供了理论指导。     

NH+4离子对重组毕赤酵母产华根霉脂肪酶发酵过程的影响

利用重组毕赤酵母在7 L的发酵罐中进行分批补料高密度发酵,考察不同NH4+质量浓度对毕赤酵母基因工程菌表达华根霉脂肪酶的影响。结果表明,补加(NH4)2SO4溶液维持NH4+质量浓度在7 g/L时华根霉脂肪酶酶活最高,分别是不补加(NH4)2SO4溶液、补加并维持NH4+质量浓度在5 g/L、10 g/L条件下的1.41、1.11、1.08倍,而且在该氮源质量浓度条件下,胞外总蛋白质质量浓度最高,达到5.61 g/L。通过分析不同氮源条件下发酵参数比生长速率、产物比形成速率和底物比消耗速率的差异,发现发酵维持NH4+质量浓度在7 g/L时,产物比形成速率和底物比消耗速率均较高,这与该条件下产酶水平最高相一致。研究结果表明,对毕赤酵母基因工程菌发酵过程中的氮源进行优化,能够明显促进外源蛋白质的表达。     

甜味蛋白Brazzein在毕赤氏酵母中表达的初步研究

目的:采用pGAP ZαA为表达载体,SMD1168毕赤酵母为受体系统,构建分泌型的甜味蛋白Brazzein毕赤酵母重组菌.方法:按照毕赤酵母偏爱密码子设计Brazzein基因,共设计4对引物.在上、下游引物分别引入Xba Ⅰ与Xho Ⅰ酶切识别位点,采用SOE-PCR法合成Brazzein基因,构建克隆载体pUC57-Bra与酵母表达载体pGAPZαA-Bra.将重组质粒线性化,电转导入毕赤酵母(Pichia. pastoris SMD1168)中,用高浓度的抗生素Zeocin筛选高拷贝转化子.将重组菌株接种于YPD培养基诱导表达,进行SDS-PAGE分析.结果:成功构建了分泌型的Brazzein毕赤酵母重组菌.SDS-PAGE表明,目标蛋白的相对分子质量与理论值一致,在诱导72h后目的蛋白表达量达0.12g/L.对纯化后的蛋白进行生物活性测定,经鉴定具有一定的甜味,表达的Brazzein蛋白生物学活性良好.     

内切葡聚糖酶基因cel7b 在里氏木霉中的同源表达

用里氏木霉（Trichoderma reesei ）作为宿主同源表达内切葡聚糖酶基因。运用聚合酶链反应（PCR）技术从里氏木霉cDNA中扩增得到内切葡聚糖酶基因cel7b序列,并将其连接到载体p18-m2上构建重组质粒,将重组质粒转化到里氏木霉菌株中,通过筛选获得表达内切葡聚糖酶的重组里氏木霉工程菌。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测显示,发酵液中重组内切葡聚糖酶的分子质量约48 ku。摇瓶发酵结果显示,内切葡聚糖酶在重组里氏木霉中得到分泌表达,发酵液中的内切葡聚糖酶和滤纸酶活分别达到了726 U/mL和28.7 U/mL,分别为出发菌株酶活的2.9倍和1.1倍。玉米芯进行糖化试验结果显示,重组里氏木霉所产酶液糖化玉米芯的酶解得率为81.4%,比出发菌株提高了6.3%。     

内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达

为实现内切葡聚糖酶和木聚糖酶在毕赤酵母中的共分泌表达,进而降低酶制剂的生产成本,构建含木聚糖酶基因的重组表达载体pPICZαA-Aoxyn11A,经SacI线性化后,电转化至含内切葡聚糖酶基因Aucel12A的重组毕赤酵母GSC7中,获得双重重组毕赤酵母GSCX8。经甲醇诱导表达后,GSCX8发酵上清液中内切葡聚糖酶和木聚糖酶的活性分别为47.77IU/mL和192.71IU/mL,为单独表达菌株GSC7和GSX5的85%和80%。酶学性质分析显示,内切葡聚糖酶的最适pH为4.0,在pH3.0~8.5稳定;最适温度为50℃,在60℃以下稳定。木聚糖酶的最适pH为5.5,在pH3.0~10.0稳定;最适温度为55℃,在50℃以下稳定。GSCX8遗传稳定性测试结果表明,内切葡聚糖酶和木聚糖酶在毕赤酵母中实现了稳定的共表达。     

Iodine-125-labeled lipoprotein lipase as a tool to detect and study spontaneous lipolysis in bovine milk

The distribution of lipoprotein lipase among cream, casein, and milk serum can be evaluated by addition of a trace amount of 125I-labeled lipoprotein lipase to milk. Radioactive lipase was distributed in parallel to endogenous lipase under several conditions. In some milk samples, binding of lipase to cream increased when the milk was cooled. Correlation was good between bound labeled lipase and degree of cold-induced lipolysis in corresponding milk samples. Binding of lipase to cream or to casein was not saturable by addition of two-to threefold more lipase than is normally present in milk. In milk with a relatively high fraction of lipase bound to cream, a correspondingly lower fraction was associated with casein, whereas the fraction of lipase in milk serum was similar in all milk samples. Cold-induced binding of lipoprotein lipase to cream was not fully reversed when the milk was warmed again. Heparin released lipase from casein and increased the amount of lipase bound to cream after cooling.     

用硅藻土固定化脂肪酶及酶法合成单甘油酯

本文研究了一种简单的用硅藻土固定猪胰脂肪酶的方法。选择合适的pH、酶量及温度 ,脂肪酶固定量可达 10 2 0U/g。用于棕榈油甘油解合成单甘油酯时 ,固定化酶热稳定性比游离酶好 ,使用寿命长 ,具有一定的应用潜力     

偏甘油酯脂肪酶的研究进展及应用现状

相较于甘油三酯脂肪酶,偏甘油酯脂肪酶因其独特的底物选择性和催化高效性,逐渐成为相关领域的研究热点。旨在为偏甘油酯脂肪酶在油脂加工、脂质分子的定向合成等领域的研究提供理论和技术参考,并为挖掘新型的偏甘油酯脂肪酶打下基础,首先对偏甘油酯脂肪酶的来源和分类进行介绍,随后阐述了偏甘油酯脂肪酶的结构特征和催化特性,最后对其应用现状进行了总结归纳和展望。偏甘油酯脂肪酶有动物来源、植物来源和微生物来源,分为甘油单酯脂肪酶和甘油单酯-甘油二酯脂肪酶;偏甘油酯脂肪酶有帽区、催化三联体和氧负离子洞穴等结构,具有底物特异性和位置特异性;偏甘油酯脂肪酶在食品、有机合成、医疗等领域有着广泛的应用和独特的发展潜力。     

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.     

有机溶剂和混合油脂对脂肪酶活性恢复的影响

本文通过多次催化酯交换方法,研究了最优催化条件下脂肪酶活性和结构的关系,以及低活性脂肪酶的活性再生技术.在催化合成1,3-二油酸-2-棕榈酸甘油三酯的研究中,利用荧光光谱和傅里叶变换红外光谱,分析最优条件下不同使用次数脂肪酶的三级与二级结构,研究脂肪酶活性变化与其二级、三级结构之间的关系.使用异丙醇和混合油脂处理脂肪酶...     

各类微生物脂肪酶酶学性质及应用研究进展

微生物脂肪酶作为一类功能强大的催化剂,越来越受到人们的关注。微生物脂肪酶种类繁多,广泛存在于霉菌、酵母和细菌中,且不同微生物来源的脂肪酶其酶学性质、基因结构各不相同。对目前国内外研究较多的根霉脂肪酶、曲霉脂肪酶、青霉脂肪酶、毛霉脂肪酶、地霉脂肪酶、酵母脂肪酶和细菌脂肪酶的最适作用温度、最适pH及pH稳定性、底物特异性、酶活性影响、基因结构及其应用等方面进行了综述，并论述了食品加工业中脂肪酶的研究进展。     

Within-day variation of lipolytic activity in human milk

Milk was collected from six mothers during their 8th wk of lactation to determine the diurnal variation of bile salt-stimulated lipase and serum-stimulated lipase. On the day of collection, one breast was completely emptied at 0600, 1000, 1400, 1800, and 2000 h. Activities of bile-stimulated lipase and serum-stimulated lipase in the milk were determined. Activity of bile-stimulated lipase was constant throughout the day, whereas serum-stimulated lipase activity varied significantly with time. Both activities differed significantly in milk from different women. Time of collection must be considered when measuring activity of serum-stimulated lipase but is not a major variable when measuring activity of bile-stimulated lipase in human milk. These results may be helpful when designing future studies involving these enzymes.     

枇杷幼果总黄酮提取物对脂肪酶的抑制效应及机制

目的研究天然提取物枇杷幼果总黄酮对脂肪酶活性的抑制效应。方法采用抑制率、抑制动力学以及荧光淬灭等方法研究枇杷幼果总黄酮对脂肪酶的作用。结果枇杷幼果总黄酮对脂肪酶有较为显著的抑制作用,半抑制浓度为(54.51±4.07)μg/mL,以竞争性与非竞争性相混合的方式抑制脂肪酶活性,且枇杷幼果总黄酮能抑制脂肪酶的荧光强度。结论枇杷幼果总黄酮提取物能与脂肪酶结合,进而抑制脂肪酶的活性。     

Glycerol as a carbon source for lipase production by the fungus rhizopus delemar

The ability of the fungus Rhizopus delemar to synthesize lipase (glycerol ester hydrolase, E.C. 3.1.1.3) when grown on glycerol as the prime carbon source was investigated. Glucose, glycerol and olive oil all supported fungal growth and lipase production. Maximum net and specific lipase activities obtained in glycerol were greater than those obtained in glucose and olive oil, and were reached earlier and maintained longer during growth. In glucose media, lipase activity did not appear until the glucose had been consumed. Differences in lipase activity between glycerol and glucose media were not due to pH‐mediated lipase inactivation. In both glucose and glycerol media the appearance of lipolytic activity coincided with the appearance of lipase polypeptide. The data are consistent with the postulate that lipase synthesis is subject to catabolite repression by glucose. These studies identify a simple, single‐phase medium for lipase production and establish the utility of glycerol for amplifying enzyme levels.     

磁性壳聚糖微球固定化脂肪酶研究

以磁性壳聚糖微球为载体,通过戊二醛交联进行脂肪酶固定化,对影响脂肪酶固定化各种因素进行考察,确定最佳条件,并比较游离酶与固定化酶pH和热稳定性。结果表明,固定化适宜条件为:脂肪酶加入量5.0 mg/100 mg载体、温度40℃、时间5 h、pH 8.04、戊二醛浓度10%、最高固载率可达90.56%,酶活4034 U/g载体;与游离酶相比,固定化酶pH和热稳定性都有较宽适用范围。