微生物脂肪酶应用研究进展

微生物脂肪酶是一种重要的工业酶类,也是当前国内外的研究热点。对脂肪酶在食品工业、有机合成、生物柴油合成、手性药物合成等方面的应用研究近况进行了综述,并进行了展望。     

添加外源脂肪酶对瓦氏黄颡鱼的生长、消化酶及血清生化指标的影响

为考察添加脂肪酶酶制剂对瓦氏黄颡鱼生长、消化酶以及血清生化指标的影响,在基础饲料中分别添加0mg/kg、100mg/kg、300mg/kg、500mg/kg脂肪酶制剂,饲喂平均体重(3.47±0.05)g的瓦氏黄颡鱼90d。试验结果表明:添加脂肪酶100mg/kg、300mg/kg、500mg/kg组分别比对照组增重率提高3.86%(P>0.05)、9.29%(P<0.05)、6.75%(P>0.05),饲料系数降低了3.17%(P>0.05)、6.35%(P<0.05)、5.29%(P<0.05)。同时添加脂肪酶各组的血清甘油三酯、低密度脂蛋白和总胆固醇含量都显著低于对照组(P<0.05),通过消化酶测定表明,脂肪酶添加各组均显著提高了胰蛋白酶和肠淀粉酶活性(P<0.05),100mg/kg脂肪酶组还显著提高了胰脂肪酶和胰淀粉酶(P<0.05),300mg/kg脂肪酶组胰脂肪酶活性最高(P<0.05)。上述研究表明,脂肪酶在瓦氏黄颡鱼饲料中的适当添加量为300mg/kg为宜。     

脂肪酶法催化胆固醇合成胆固醇棕榈酸酯的研究

探讨了固定化Candida antarctica脂肪酶催化胆固醇与棕榈酸合成胆固醇棕榈酸酯的反应过程。通过脂肪酶催化酯化反应的工艺路线进行研究,系统考察了转速、反应介质、醇酸比、固定化酶用量、反应温度等对酯化反应的影响。结果表明,以环己烷为溶剂,在30℃,胆固醇与棕榈酸物质的量比1：1,2%（质量分数）脂肪酶,反应时间为48h的条件下,反应的最高酯化率可达83%以上,产品经HPLC和LC-MS分析的纯度可达98%以上;固定化C.antarctica脂肪酶具有较高的催化稳定性,其半衰期至少达120d。     

饲料中去皮豆粕替代鱼粉比例对草鱼消化酶活力的影响

试验旨在研究去皮豆粕替代鱼粉比例对草鱼消化酶活力的影响。选择健康的草鱼为试验对象,以鱼粉为动物蛋白源,去皮豆粕为植物蛋白源,去皮豆粕分别替代0%、15%、30%、45%和60%的鱼粉蛋白,配制成5种等蛋白、等能的半精制饲料,在室内单循环控温养殖系统中进行8周生长试验。试验结束后,取草鱼的前、中、后段肠道和肝胰脏,分别用福林-酚试剂法、碘-淀粉比色法和脂肪酶试剂盒测定了肠道和肝胰脏蛋白酶、淀粉酶和脂肪酶的活力。结果表明:饲料中去皮豆粕替代鱼粉对草鱼肠道和肝胰脏的蛋白酶活力具有显著影响,随着去皮豆粕替代鱼粉蛋白水平的增加,草鱼肝胰脏和肠道蛋白酶活力逐渐降低,60%组与对照组差异显著(P0.05);去皮豆粕替代鱼粉对草鱼肝胰脏、前肠、后肠淀粉酶活力无负面影响,但草鱼中肠淀粉酶活力45%和60%组显著低于对照组(P0.05);去皮豆粕替代鱼粉对草鱼肝胰脏和肠道脂肪酶活力影响不显著(P0.05)。     

基因组步移技术扩增嗜水气单胞菌J-1株脂酶基因全长及原核表达

根据已发表的引物序列合成一对引物,以嗜水气单胞菌J-1株基因组为模板扩增脂酶基因,得到一个保守的383 bp基因片段。进一步应用基因组步移技术扩增其两侧未知序列,分别以DraⅠ,EcoRⅤ,PvuⅡ,ScaⅠ,StuⅠ,SmaⅠ6个内切酶酶切的基因组构建6个不同的基因组步移文库,再以基因组步移文库为模板,设计引物向已获得的保守片段两侧进行PCR扩增,将扩增到的两侧序列与保守片段拼接后得到一个3 476 bp的片段,通过DNA Star软件分析,找到一个2 415 bp的开放阅读框,经Blast软件分析,其与嗜水气单胞菌ATCC 7966脂酶、嗜水气单胞菌AH-3磷脂酶A1、嗜水气单胞菌H3脂酶、嗜水气单胞菌Mcc-2脂酶、嗜水气单胞菌J MP636磷脂酶C的序列同源性分别为97%、97%、88%、83%和82%。将DNA序列翻译成氨基酸序列后,发现其包含一个多肽序列(VHFLGHSLGA),这个序列在脂酶中高度保守。     

立体分子伴侣-脂肪酶特异折叠酶

细菌脂肪酶是一类重要的工业用酶,尤其以来源于假单孢杆菌Pseudomonas与伯克霍尔德菌Burkholderia的脂肪酶应用最为广泛。然而,这类脂肪酶折叠过程中存在一个巨大的能障而无法自发正确折叠。研究发现,这类脂肪酶的编码基因所在操纵子中往往存在一个与其折叠相关的基因,编码一种脂肪酶特异折叠酶。该类蛋白为脂肪酶的立体分子伴侣,可与脂肪酶的折叠中间体相互作用,帮助脂肪酶越过折叠过程中的能量障碍,为其获得正确构象提供必要的空间信息。详细阐述了脂肪酶特异折叠酶的发现、分类、作用机制与应用前景。     

Anti-diabetic effects of emodin involved in the activation of PPARγ on high-fat diet-fed and low dose of streptozotocin-induced diabetic mice

Rheum palmatum Linn has been widely applied in the clinical treatment of diabetes mellitus. It has been found that emodin as the major bioactive component of R. palmatum L exhibits the competency to activate peroxisomal proliferator-activated receptor-γ (PPARγ) in vitro. So the aim of this study was to evaluate the anti-diabetic effects of emodin through the activation of PPARγ on high-fat diet-fed and low dose of streptozotocin (STZ)-induced diabetic mice. The diabetic mice were intraperitoneally injected with emodin for three weeks. No changes of food consumption and the body weight in emodin-treated mice were monitored daily during the entire experiment. At the end of experiment, the levels of blood glucose, triglyceride and total cholesterol in serum were significantly decreased after emodin treatment. However, serum high-density lipoprotein cholesterol (HDLc) concentration was significantly elevated. The glucose tolerance and insulin sensitivity in emodin-treated group were significantly improved. Furthermore, the results of quantitative RT-PCR analysis showed that emodin significantly elevated the mRNA expression level of PPARγ and regulated the mRNA expressions of LPL, FAT/CD36, resistin and FABPs (ap2) in liver and adipocyte tissues. No effects on the mRNA expressions of PPARα and PPARα-target genes were observed. Taken together, the results suggested that the activation of PPARγ and the modulation of metabolism-related genes were likely involved in the anti-diabetic effects of emodin.     

不同烟草类型种子萌发期脂肪酶及脂类物质动态研究

以烤烟K 326和白肋烟TN 86为材料,研究了烟草种子萌发过程中脂肪酶活性与脂类物质含量动态。结果表明,无论是白肋烟,还是烤烟,脂肪酶活性与脂肪酸含量都随种子萌发进程呈上升趋势,其间有所波动,但彼此间动态极显著正相关,脂肪酶活性高值期在96 h,脂肪酸含量高值期在168 h,脂肪酸含量高值期滞后于脂肪酶活性高值期。烟草种子中粗脂肪含量较高,60 h前变化甚微,60 h后呈下降态势,至萌发结束(336 h)时值最低,其含量动态与脂肪酸呈极显著负相关。萌发前及萌发期间白肋烟的脂肪酶活性和脂肪酸含量大多高于烤烟,而粗脂肪含量则又是烤烟高于白肋烟,不同烟草类型间差异较明显。     

瓦氏黄颡鱼肝脂酶基因cDNA序列的克隆与序列分析

利用RT-PCR方法克隆瓦氏黄颡鱼(Pelteobagrus vachelli)肝脂酶cDNA序列片段,并对其基因结构和系统进化关系进行分析.瓦氏黄颡鱼肝脂酶cDNA片段长1065 bp,编码353个氨基酸.肝脂酶氨基酸序列同源性分析结果表明,瓦氏黄颡鱼与其他鱼类的同源性为62%-100%.瓦氏黄颡鱼肝脂酶氨基酸残基包含糖基化位点、催化位点、催化中心三联体位点、脂质结合位点和多肽"盖"等主要结构区域.系统进化树分析表明,瓦氏黄颡鱼肝脂酶与草鱼、鳙、斑马鱼肝脂酶聚为一支.因此,瓦氏黄颡鱼的肝脂酶在鱼类进化中较为保守.     

Fucoidan from marine brown algae inhibits lipid accumulation

In this study, we elucidated the inhibitory effect of fucoidan from marine brown algae on the lipid accumulation in differentiated 3T3-L1 adipocytes and its mechanism. The treatment of fucoidan in a dose-dependent manner was examined on lipid inhibition in 3T3-L1 cells by using Oil Red O staining. Fucoidan showed high lipid inhibition activity at 200 μg/mL concentration (P < 0.001). Lipolytic activity in adipocytes is highly dependent on hormone sensitive lipase (HSL), which is one of the most important targets of lipolytic regulation. Here, we examined the biological response of fucoidan on the protein level of lipolysis pathway. The expressed protein levels of total hormone sensitive lipase (HSL) and its activated form, phosphorylated-HSL were significantly increased at concentration of 200 μg/mL fucoidan. Furthermore, insulin-induced 2-deoxy-D-[3H] glucose uptake was decreased up to 51% in fucoidan-treated cells as compared to control. Since increase of HSL and p-HSL expression and decrease of glucose uptake into adipocytes are known to lead to stimulation of lipolysis, our results suggest that fucoidan reduces lipid accumulation by stimulating lipolysis. Therefore, these results suggest that fucoidan can be useful for the prevention or treatment of obesity due to its stimulatory lipolysis.