超声波对猪胰脂肪酶催化活性影响的机理研究

猪胰脂肪酶(porcine pancreas Lipase,PPL)反应系统经适宜参数的超声波处理,催化活性提高了11.22%～24.42%。PPL经超声波处理后,其动力学参数Km和Vmax都变小,酶分子的紫外吸收光谱不变,荧光发射光谱不变。而酶分子经超声波处理后,其紫外差示光谱出现了明显的正峰或负峰。探讨了超声波影响PPL活性的作用机理。     

海泡石吸附固定化猪胰脂肪酶

以海泡石作为猪胰脂肪酶(PPL)的固定化载体,考察采用物理吸附的方法制备固定化脂肪酶的条件。结果表明:在固载时间4 h、反应磷酸盐(PBS)溶液pH 6.0、反应温度25℃时,可达最大比酶活309 U/g,固定化酶的化学稳定性和热稳定性均较高。同时利用红外谱仪(FT-IR)和扫描电子显微镜(SEM)的分析手段对固定化猪胰脂肪酶试样进行分析,进一步确定了海泡石材料在固定化酶中的作用。     

功能化离子液体化学修饰猪胰脂肪酶的催化性能

选取氯化1-羧甲基-3-甲基咪唑、氯化1-羧甲基-3-乙基咪唑、氯化1-羧甲基-3-丁基咪唑3种离子液体对猪胰脂肪酶(PPL)进行化学修饰,得到3种修饰的脂肪酶分别命名为PPL-M、PPL-E、PPL-B。以三乙酸甘油酯水解为模型反应,考察离子液体修饰前后PPL的活力、热稳定性、耐有机溶剂性等酶学性质,并通过紫外光谱研究修饰对PPL空间结构的影响。结果表明:修饰后PPL的活力明显提高,对温度和pH的敏感度降低。修饰酶的热稳定性明显提高,在高浓度的甲醇及N,N-二甲基甲酰胺(DMF)中仍能保持游离酶活力的100%。修饰后酶的特征吸收峰发生红移,吸收强度增强,修饰后酶的微环境发生了改变。     

纳米银复合介孔碳材料制备固定化猪胰脂肪酶

以稻壳为原料制备介孔碳材料,对其表面进行纳米银修饰后作为猪胰脂肪酶(PPL)的固定化载体.初步研究并优化该载体固定猪胰脂肪酶的条件.结果表明:在AgNO3浓度为0.01 mol/L、固载时间6h、反应磷酸盐溶液pH为6.0、反应温度25℃时,可达最大酶活,酶活提高3.2倍.同时利用红外图谱(FT-IR)、N2等温吸附-脱附曲线(BET)和扫描电子显微镜(SEM)的分析手段对固定化猪胰脂肪酶试样进行分析,进一步确定了纳米银复合介孔性碳材料在固定化酶实验中的作用.     

脱脂棉固定化脂肪酶的非水活性与稳定性

脂肪酶具有非水催化作用,但其非水催化活性和稳定性需进一步提高,这是非水酶学的瓶颈问题之一。理想的策略是模拟脂肪酶的界面活化机制,以大分子代替水,优化、稳定化和有效分散酶蛋白,阻止其在有机相中变性。因此,选用多羟基、比表面积大、惰性、且与酶蛋白能兼容的大分子--脱脂棉纤维,作为固定化载体,以1∶0.9的质量比,通过物理吸附,将假单胞菌脂肪酶(Pseudomonas cepacia lipase)固定在脱脂棉纤维上。在催化己醇与乙酸乙烯酯的转酯反应中,反应1 h,脱脂棉固定化脂肪酶转化底物的能力是酶粉的3.7倍。在每次6 h共6次的循环催化中,固定化酶和酶粉转化底物的能力分别平均每次降低约0.3%和2.4%。表明脱脂棉固定化脂肪酶的非水活性,尤其是稳定性明显提高。这为通过固定化有效提高脂肪酶的非水催化作用,以满足工业应用的需要,提供了一种有效的途径和重要参考。     

脂肪酶活力测定方法及其在筛选产脂肪酶微生物中的应用

比较了罗丹明平板法、橄榄油乳化法、对硝基苯酚法测脂肪酶水解酶活和对硝基苯酚法测脂肪酶酯合成酶活4种常用的脂肪酶活力测定方法。结果表明,罗丹明平板法只适合脂肪酶活力的定性和初步定量判断,后3种方法适合于脂肪酶活力的定量检测,但只有对硝基苯酚法有较好的重现性。而且,脂肪酶水解酶活力和其合成活力无对应关系,所以在筛选产脂肪酶微生物时要选择合适的脂肪酶活力测定方法。也即,筛选产水解酶活的菌株应选择水解酶活测定方法,否则,选择酯合成酶活力测定方法。基于这一原则筛选到了预期产脂肪酶微生物。     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

脂肪酶及其在化学品合成中的应用

脂肪酶催化过程具有高效和高选择性、条件温和以及环境友好等特点。目前可再生能源和绿色化工领域对新型酶催化转化技术的迫切需求使得越来越多的脂肪酶被应用到生物柴油、精细化学品和医药中间体合成的领域。本文主要介绍了脂肪酶的催化技术及其在化学品合成中的应用。     

纳米载体固定化脂肪酶及其在生物柴油转化中的应用进展

随着全球能源需求量的不断上升和日益加剧的环境压力,固定化脂肪酶在可持续生物柴油合成中的应用受到广泛关注。纳米材料,包括纳米粒子(磁性和非磁性)、碳纳米管和纳米静电纺丝,具有比表面积大、结构稳定、易于功能化修饰等优势,是固定化脂肪酶领域的重要载体之一。综述了纳米材料作为载体在脂肪酶固定化中的应用,重点介绍这类生物催化剂在生物柴油合成中的最新进展,并对纳米材料固定化脂肪酶发展前景进行展望,旨在为固定化脂肪酶的研究和工业化应用奠定基础。     

大孔吸附树脂固定猪胰脂酶的初步研究

目的：为促进脂肪酶在工业上的应用,对以大孔吸附树脂为载体的猪胰脂酶固定进行了研究。方法：以吸附法固定,用单因素法考察了吸附条件对固定的影响。按Eadie—HOfstee法测定了固定化酶及游离酶的表观米氏常数,并具体研究了固定化酶的操作稳定性、热稳定性等性质。结果：固定化酶的比酯交换能力比游离酶上升6倍;最优的吸附条件为：pH7．0,酶加量300mg/g,水分含量20％;吸附时间4h;固定化酶在含水量10％时可稳定操作5批次;同时固定化酶有比游离酶有更好的热稳定性。结论：以大孔吸附树脂为载体对猪胰脂酶进行固定是可行的;与游离酶相比,所得固定化酶有着更高的比酯交换能力和更好的稳定性,更为适用于工业化生产。     

脂肪酶在反相胶囊中的催化行为的研究

系统研究了脂肪酶在ＡＯＴ／水／异辛烷反应相胶囊中的催化行为。在一定条件下,反相胶囊中的酶反应的仍符合Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学原理,研究了含水量,底物浓度,ｐＨ,温度,溶剂的种类和表面活性剂浓度等对酶反应的影响。结果表明,酶活力与Ｒ值（水与表面活性剂的摩尔比值）有关。获得最大酶活力的条件是Ｒ＝１１,ｐＨ７．０,温度３２．５℃,橄榄油浓度为４０％。     

Lipase Activity of Mucor pusillus

Two strains of Mucor pusillus were examined for their ability to synthesize lipase in a complex medium used in the production of milk-clotting protease. Lipase activity of both strains reached maximal after 6 days of incubation under submerged conditions at 35 C. Lipase secreted into the medium hydrolyzed butterfat and vegetable lipids, as well as selected synthetic triglycerides. About 50% of lipase activity was destroyed after a 45-min heat treatment at 58 C.     

Variation of Telomerase Activity and Morphology in Porcine Mesenchymal Stem Cells and Fibroblasts during Prolonged in vitro Culture

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-ß-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P < 0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P < 0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of ß-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0 / G1 phase was significantly (P < 0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.     

猪胰脏羧肽酶转酯功能和耐酸抗蛋白酶特性研究

羧肽酶(carboxypeptidase)是一类可水解肽链C末端氨基酸残基的蛋白酶,可用作饲料添加剂,促进畜禽生长。猪胰脏中含有一种羧肽酶A1(CPA1),该酶除能水解蛋白质外,还能分解油脂。CPA1在以酪蛋白和橄榄油为底物时,比活力分别为53.14 U/mg和22.4 U/mg;经过p H2.0酸性环境和胰蛋白酶处理后,其酶活残留率分别为95.65%和78.14%。结果表明,CPA1具有转酯功能和耐酸抗蛋白酶特性。     

Demonstration of Activity of Gastrin in Mammalian Pancreas

THE gastrin peptides are now known to be the hormonal mediator of the Zollinger–Ellison (ZE) syndrome¹, but the search for a non-insulin, non-histamine and/or a gastrin-like secretagogue in the normal pancreas has yielded conflicting results. Hallenbeck et al.² and Lai³, using bioassay procedures, failed to detect gastrin activity in extracts of normal pancreas from a variety of mammals including man, hog and monkey, but in man⁴ and in pig, panther, man, rabbit, albino rat and guinea-pig⁵ gastrin-containing cells have been detected by antigastrin immunofluorescence in normal pancreatic tissue. The discrepancy between the results of extraction/bioassay and immunofluorescence detection could be due either to the presence of an immunoreactive but biologically inactive gastrin deptide or its precursor in the normal pancreas, or the failure to extract and/or detect by the extraction and assay procedures used the biologically active gastrin present perhaps in very small amounts in the normal pancreas.     

Lipase Activity in Germinating Seedlings of Cucumeropsis edulis

Acetone powders prepared from dormant and germinating seedlingsof Cucumeropsis edulis contained an active lipase system whichshowed increasing lipase activity with germination. Kineticproperties indicated maximum activity at pH 5 and at a temperatureof 37 ° C, and the enzyme exhibited a zero-order reactionrate for the first 2 h of hydrolysis. The results obtained showedthat the enzyme is non-specific in its mode of action, hydrolysingfatty acids irrespective of chain length, degree of unsaturation,and the position of fatty acids in the triglycerides. The deterioration and spoilage of ‘ground’ seedsin storage has been shown to be due to the release of endogenouslipase during grinding. The release of lipase is greater thegreater the water content of the seeds     

Determination of Lipase Activity in AOT-Isooctane Reversed Micelles

In fission yeast, the conserved proteins, MO25/Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.