甘三酯立体专一分析的研究：（Ⅱ）甘三酯立体专一分析方法初探

在第一部分综合讨论的基础上对甘三酯立体专一分析方法作了初步探讨。以液体油(菜油)及固体脂(猪脂)为基质,系统地研究了这一分析方法,取得了经验并补充了一些具体验证方法。分析液体油的结果与文献数据相一致,分析猪脂的结果欠佳,还存在一些问题需待进一步研究。     

Link between Lipid Second Messengers and Osmotic Stress in Plants

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.     

磷脂酶用于大豆油脱胶的研究

植物油的酶法脱胶是一种新的大豆油脱胶方法。利用新型微生物磷脂酶Lecitase Ultra进行大豆油脱胶的研究,探讨了若干操作参数对大豆油脱胶效果的影响,确定了该酶较优的反应条件:反应时间200 min,加酶量25 mg/kg,pH值4.8,温度46~48℃,大豆油含磷量能降到4.7 mg/kg。结果表明,Lecitase Ultra应用于植物油脱胶效果好且稳定,是一种更适宜于工业化应用的酶种。     

粘质沙雷氏菌PL-06磷脂酶A1在大肠杆菌中的分泌表达及其条件优化

克隆粘质沙雷氏菌PL-06磷脂酶A1基因pla A,与载体p ET-22b（+）连接构建重组质粒p ET-22b（+）-pla A,并将重组质粒导入受体菌E.coli BL21（DE3）中表达,构建得到重组菌AP22,IPTG诱导表达目的蛋白后将培养基蛋白进行SDS-PAGE分析显示:重组蛋白以可溶性蛋白的形式大量存在于发酵液中,分子质量约35 ku,与预期蛋白大小一致。以磷脂酶A1酶活为指标,在单因素实验的基础之上,通过正交实验得到摇瓶培养最佳条件为:氨苄青霉素终浓度30μg/m L,IPTG加量0.25 mmol/L,温度为34℃,OD600值为0.3,诱导时间4 h。在此条件下重组胞外磷脂酶A1酶活可高达8.6 U/m L,比优化前提高了43.3%。      

磷脂酶研究进展

磷脂酶是在生物体内存在的可以水解甘油磷脂的一类酶,其中主要包括磷脂酶A1、A2、B、C和D,它们特异地作用于磷脂分子内部的各个酯键,形成不同的产物,被广泛用于甘油磷脂的改造。依靠传统手段从动物脏器中提取的磷脂酶已经不能满足目前生产的需求,而微生物来源磷脂酶的筛选及其基因工程发展迅速,显著拓宽了磷脂酶的来源,同时又有效地改善了磷脂酶的性质。本文综述了近年磷脂酶高产微生物菌株的选育、磷脂酶的分子改造以及修饰方面取得的进展。      

磷脂酶C对大豆原油脱胶效果的研究

为了避免传统脱胶方式引起的化学品消耗大、水耗高,而且脱胶油得率低、残磷量高等问题,以大豆原油为原料,采用磷脂酶C对其进行酶法脱胶,考察加酶量、柠檬酸添加量、脱胶温度和脱胶时间对磷脂脱除和脱胶油得率的影响。结果表明,磷脂酶C脱胶的最佳反应条件为0.45 g/mL柠檬酸添加量0.15 mL(100 g原油)、加酶量40 mg/100 g(以油质量计)、脱胶温度50℃、脱胶时间4 h。在最佳条件下,磷脂酶C脱胶油中含磷量降至1.00 mg/kg。酶法脱胶油的脂肪酸组成和各脂肪酸相对含量与大豆原油相比无显著差异。与酸化脱胶相比,酶法脱胶油的得率无显著差异,油中甘一酯和甘二酯相对含量分别增加了0.49、1.15百分点,甘三酯相对含量减少了2.28百分点,油脚中磷脂的组成及各组分相对含量无变化。     

磷脂酶A1水解蛋黄卵磷脂的研究

采用磷脂酶A1水解蛋黄卵磷脂,通过单因素实验和正交实验确定该酶水解蛋黄卵磷脂经济合理的工艺条件为：反应温度50℃,反应时间5h,加酶量0．40 IU／g,起始pH6．0,底物质量浓度80g／L;利用电喷雾飞行时间质谱仪分析水解前后蛋黄卵磷脂组分,磷脂酶A1水解Sn-1位脂肪酸,生成含Sn-2位不饱和脂肪酸的溶血磷脂,反应过程中有酰基位移发生。     

磷脂酶A2用于菜籽油脱胶

利用磷脂酶A2对菜籽油进行脱胶工艺研究,采用正交实验对磷脂酶脱胶的工艺条件进行了优化。结果表明,酶用量90μL/kg,pH6.0,含水量2%,反应温度40℃,反应时间3 h,最终含磷量小于10 mg/kg,达到了理想的脱胶效果。     

Hepatic patatin-like phospholipase domain-containing protein 3 sequence,single nucleotide polymorphism presence,protein confirmation,and responsiveness to energy balance in dairy cows

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), commonly known as adiponutrin, is part of a novel subfamily of triglyceride lipase enzymes with potential effects on triglyceride metabolism in adipose and hepatic tissues. The predicted bovine PNPLA3 sequence has been identified, but expression of the gene had not been examined. The objectives of this study were to confirm the predicted bovine PNPLA3 gene sequence, determine expression of the bovine PNPLA3 gene in response to whole-animal energy balance, identify single nucleotide polymorphisms present in dairy cows, and verify the presence of the protein in the liver. Using liver biopsy samples collected from cows at +28 d relative to calving (DRTC), RNA was isolated and used to generate a cDNA template for amplification of the entire predicted coding sequence of PNPLA3 via PCR. To determine if energy balance alters the expression of PNPLA3, RNA was isolated and mRNA expression quantified in liver samples from mid-lactation cows after a 5-d ad libitum period (n = 5) and after a subsequent 5-d 50% feed restriction period (n = 5), and in samples collected from cows at −14, +1, +14, and +28 DRTC (n = 16). The presence of PNPLA3 protein was detected by Western blot in liver protein samples collected at +28 DRTC. Expression of hepatic PNPLA3 was decreased after a period of feed restriction (8.14 vs. 1.08 ± 2.17 arbitrary units, ad libitum vs. fasted). Expression of PNPLA3 mRNA was decreased at +1 and +14 DRTC compared with −14 DRTC (23.35, 7.28, 10.17, and 14.5 ± 4.9 arbitrary units, −14, +1, +14, and +28 DRTC, respectively). The presence of PNPLA3 protein was detected as a 55-kDa band in hepatic protein isolations from liver tissue collected at +28 DRTC. These data confirm the presence and sequence of the bovine hepatic PNPLA3 gene and single nucleotide polymorphisms. Furthermore, these data indicate responsiveness of bovine hepatic PNPLA3 to energy balance.     

色褐链霉菌磷脂酶D基因pld的克隆与表达

利用表达载体pET-22b( ),实现了色褐链霉菌磷脂酶D基因在大肠杆菌BL21(DE3)中的高效表达.利用镍亲和柱对表达产物进行纯化,将纯化后的重组磷脂酶D作用于底物卵磷脂和丝氨酸定向合成磷脂酰丝氨酸,并用HPLC法检测酶活力.结果表明,目的蛋白可在短时间内进行大量表达.转酯反应6 h后卵磷脂的转化率达到31%,重组磷脂酶D活力达到39 U·(mg蛋白)-1.