聚丙烯腈膜固定化海藻糖合成酶的研究

以聚丙烯腈膜(PAN)为载体,采用吸附法固定化海藻糖合成酶粗酶液。通过单因素法探讨最佳固定化条件,并分析固定化酶酶学性质。结果表明,最佳固定化条件为:pH 7.6、30℃、加酶量0.1 mg/cm~2条件下震荡吸附3 h;该条件下制备的固定化酶最适反应条件为:温度40℃、pH 7.4、初始麦芽糖底物浓度200 g/L。与游离酶相比,固定化酶热稳定性提高10℃、酸碱稳定性由pH 6.6～7.4扩展至pH 5.4～8.0;反应达到平衡时,反应液中海藻糖含量为50.9%,副产物葡萄糖含量为9.1%,较游离酶降低6个百分点,在目前已报道的固定化海藻糖合成酶催化反应中副产物含量最低;在40℃、pH 7.4、麦芽糖底物浓度200 g/L条件下,重复使用累计72 h后,固定化酶活仍保留在初始酶活的64.4%。     

纤维素膜固定化麦麸酯酶的工艺研究

以麦麸中的植物酯酶为酶源,通过交联、吸附方法比较,选择物理吸附法将其固定于玻璃纤维素膜上,以酶活回收率为指标,探索其固定化的最佳条件。在单因素试验的基础上,利用Box-Behnken试验,进一步研究了缓冲液pH值、固定化时间、固定化温度3个因素及交互作用对麦麸酯酶固定化效果的影响,确定最佳固定化条件为:磷酸盐缓冲液pH值7.0,固定化时间4h,固定化温度29℃,该条件下酶活回收率的预测值为28.21%,验证值为27.51%。该研究为利用固定化麦麸酯酶快速检测有机磷和氨基甲酸酯类农药残留提供了技术参考。     

聚丙烯-TiO2/CA复合膜固定化脂肪酶最佳条件的研究

以聚丙烯-TiO2/CA复合膜对脂肪酶进行吸附固定,研究了最佳固定化条件:温度20℃,pH8.0,振荡速度100 r/min,吸附时间2h,膜酶活力最高为4.5U/cm2。膜固定化酶转化反应最佳条件:温度35℃,比游离酶降低了5℃;最适pH 8.5,与游离酶相比pH向碱性偏移,间歇水解橄榄油132h酶活为原酶活的56.7%。     

对苯醌活化法固定化脂肪酶的研究

研究了对苯醌活化法在纤维素滤纸膜载体上固定化猪胰脂肪酶的最佳条件。结果表明,当对苯醌浓度为０．０１ｇ／ｍｌ,活化６０ｍｉｎ,与浓度为０．００５ｇ／ｍｌ的酶的ｐＨ８．０的磷酸盐缓冲溶液于４℃交联２４ｈ,获得的固定化酶活最高,为０．４４Ｕ／ｃｍ＾２。固定化酶最适温度３５℃,最适ｐＨ９．０。     

不同材料固定化柚苷酶的比较

选取壳聚糖、海藻酸钠、活性炭和氧化石墨烯对柚苷酶进行固定化,比较不同材料对柚苷酶的载酶率和固定化酶活,得到较佳的固定化材料为氧化石墨烯,其载酶率达85.61%,酶活达410.50 U/g,重复使用7次后仍能保持72.38%的相对酶活。同时研究了不同条件对氧化石墨烯固定化柚苷酶催化活性影响,得到较佳的固定条件:载酶量为每克氧化石墨烯材料承载60 mg柚苷酶,固定化温度为20℃,p H值为4.0,固定化吸附时间为9 h,优化后氧化石墨烯固定化柚苷酶的酶活为433.70 U/g,重复使用7次后仍保留78.62%的酶活。     

海藻酸钠包埋法固定青霉产菊粉酶的研究

利用海藻酸钠包埋法固定青霉(penicillium)产菊粉酶,研究了最佳固定化条件及固定化酶性质,为固定化菊粉酶应用于果糖工业生产提供理论依据。结果表明:海藻酸钠浓度为4%,氯化钙浓度为1%时,固定化酶活力最高且机械强度好;加酶量越少,固定化效率越高;固定化酶与游离酶的最适pH为4.5,但固定化酶在pH4到6.5范围内相对酶活要比游离酶酶活高;固定化酶与游离酶的最适温度均为55℃,在35℃到55℃范围内两者酶活变化不大,在55℃到75℃范围内固定化酶的温度适应性比游离酶的好;在重复利用方面,将固定化酶反复利用7次,酶活仍为原酶活的50.6%。由此说明,固定化酶成本低,利用率高,在工业生产方面具有利用价值。     

鞘氨醇单胞菌产3-苯氧基苯甲酸降解酶固定化条件及其酶学性质研究

以海藻酸钠为载体,对鞘氨醇单胞菌SC-1产3-苯氧基苯甲酸(3-PBA)降解酶进行固定化,探讨酶的固定化条件及固定化酶的酶学性质。结果表明,当酶液与海藻酸钠体积比为1∶5、海藻酸钠质量分数2%、氯化钙质量分数1%时,固定化酶的酶活较高,固定化时间对酶活影响不显著,而固定化酶凝珠直径对其酶活影响较大,在最优条件下固定化酶的酶活达223.1 U/g,酶活回收率为86%。在30℃,p H 7.0时固定化酶的酶活达到最高值;Mg2+,Mn2+及较高浓度Ba2+,低浓度Cu2+,10-2~10-6mol/L EDTA,0.01%BSA,10-6mol/L邻菲罗啉,0.1%VC对固定化酶的酶活有促进作用。固定化酶的米氏常数(Km)为46.63×106nmol/L,最大反应速率(Vmax)为3.70×103U/g。固定化3-PBA降解酶在最适条件下连续反应900 min,对5μg/m L的3-PBA降解率为26.14%。     

戊二醛交联法固定化脂肪酶活性包涵体的研究

目的以戊二醛(GA)为交联剂固定化黏质沙雷菌ECU1010脂肪酶活性包涵体。方法单因素优化固定化条件,以GA浓度、酶浓度、GA与酶用量比、固定化时间4因素3水平正交试验确定最佳固定化条件,并研究固定化酶的操作稳定性和贮存稳定性。结果最佳固定化条件为:GA浓度0.15%(w/v),酶浓度15 mg/mL,GA与酶用量比6:1,固定化时间2 h。此条件下制备的固定化酶平均酶活达234.7 U/L,酶活回收率40%以上。固定化酶重复使用6次后仍能保持50%的酶活。4℃贮存3个月后其酶活基本无明显减弱。结论脂肪酶活性包涵体经固定化后其操作稳定性和贮存稳定性显著提高。     

壳聚糖／碳酸钙杂化膜固定化糖化酶的研究

以壳聚糖、碳酸钙等为载体制备杂化膜,研究杂化膜的最佳制备条件.研究结果表明,当碳酸钙含量为1.803%时,膜较柔软,有弹性,不溶解,杂化膜相对酶活最高,达74.42%.以此杂化膜固定糖化酶.对固定化酶的酶学性质研究结果表明,固定化酶的最适温度为65℃,最适pH为4.6,固定化酶的耐热性及pH稳定性较游离酶有所提高.     

柔性固定化木瓜蛋白酶的制备及在酶解波纹巴非蛤中的应用

在壳聚糖膜的表面接上分子链较长的双醛淀粉作为柔性固定化酶的载体,将木瓜蛋白酶固定在该载体上制成柔性固定化木瓜蛋白酶,并应用于酶解波纹巴非蛤制备小分子肽。通过考察温度、pH 值、加酶量和固定化时间等条件对柔性固定化木瓜蛋白酶的酶活、酶活回收率的影响,确定最优固定化条件。结果表明：双醛淀粉用量0.8mg/g 壳聚糖、柔性固定化酶温度25℃、柔性固定化酶时间20h、加酶量40mg/g 壳聚糖、pH8.0 条件下所得的固定化酶酶活最高,酶活回收率63.35%。将该柔性固定化酶在酶解温度40℃、pH7.0、加酶量1.0%、酶解时间4h条件下制备波纹巴非蛤小分子肽,产率为3.4055%。     

Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability

In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization.     

高碘酸钠氧化法固定化磷脂酶A1的研究

研究了高碘酸钠氧化法在纤维素滤纸膜载体上固定化磷脂酶A₁的最佳条件。结果表明,以固定化酶活力为指标,当高碘酸钠浓度为0.15mol/L,活化80min,与浓度为0.05g/mL酶的磷酸盐缓冲溶液（pH为5.8）于戊二醛浓度0.4%、温度4℃交联4h,获得的固定化磷脂酶A₁酶活最高为4.8U/cm²。固定化酶膜的酶学性质为:最适温度35℃;最适pH为9.0。与游离酶相比,pH向碱性偏移1.0;经7次重复使用后,固定化酶膜活力为原酶活的65%以上。SEM结果显示,经高碘酸钠氧化后的纤维素滤纸膜能较好地固定磷脂酶A₁。     

Milk lipoprotein lipase distribution in the major fractions of bovine milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored less than or equal to 24 h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72 h of cold (4 degrees C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4 degrees C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

醋酸纤维素-聚四氟乙烯复合膜固定化脂肪酶的研究

以醋酸纤维素(CA)和聚四氟乙烯(PTFE)为材料制备醋酸纤维素-聚四氟乙烯复合膜,采用吸附-交联相结合的固定化方法,用该复合膜固定化脂肪酶。研究温度、吸附时间、酶液质量浓度、交联时间和交联剂体积分数对脂肪酶固定化效率和催化效果的影响,并对固定化酶膜的酶学性质进行研究。得到最佳的固定化条件为：温度25℃、吸附时间2h、酶液质量浓度0.02g/mL、交联时间3h、交联剂(戊二醛)体积分数0.2%,固定化酶最大酶活力为17.2U/cm2。固定化酶膜的酶学性质为：最适温度35℃,比游离酶降低了5℃;最适pH8.5,与游离酶相比pH值向碱性偏移1.0;经10次(10h /次)重复使用后,固定化酶相对酶活力为55.5%。SEM结果显示CA-PTFE复合膜能较好的固定化脂肪酶。     

从萌发的油菜种子中制备脂肪酶

测定不同萌发期油菜幼苗的脂肪酶活性,采用“盐析→超滤→M-Bondapak-C18 柱层析”的工艺路线,建立一种简单且高效的油菜脂肪酶纯化方法。结果表明：通过测定不同培养时间的油菜幼苗的脂肪酶比活力,发现培养6d 的油菜幼苗脂肪酶比活力最高,此时期的油菜幼苗适合作为脂肪酶提取的材料;对脂肪酶比活力为1.89U/mg的油菜蛋白粗提物采用盐析法沉淀脂肪酶时,得到脂肪酶的比活力为2.07U/mg,回收率为82.47%;采用截留分子质量为10000D 的超滤膜对脂肪酶盐析物进行脱盐和除去小分子杂蛋白处理,得到的脂肪酶的比活力为2.46U/mg,回收率为75.84%;进一步采用M-Bondapak-C₁₈ 柱层析纯化超滤液,脂肪酶最高比活力为9.14U/mg,回收率为34.13%。所得到的脂肪酶的活性达到了商品化脂肪酶的水平。     

Lipase localization in Rhizopus oryzae cells immobilized within biomass support particles for use as whole-cell biocatalysts in biodiesel-fuel production

To identify the lipase responsible for the methanolysis activity of fungus whole-cell biocatalysts, the lipase localization of Rhizopus oryzae cells was determined. Western blot analysis showed that R. oryzae cells produce two types of lipase with different molecular masses of 34 and 31 kDa; the former (ROL34) was bound to the cell wall, whereas the latter (ROL31) was mainly bound to the cell membrane. It was found that cell immobilization within reticulated polyurethane foam biomass support particles strongly inhibits the secretion of membrane-bound lipase into the culture medium. An investigation of the relationship between ROL34 and ROL31 suggested that ROL31 originates from the cleavage of a 28-amino-acid residue at the N-terminus of ROL34. The addition of olive oil to the culture medium led to the retention of increased amounts of lipase within the cell. This phenomenon was further confirmed by an immunofluorescence labeling of hyphal cells. When cells were cultivated with various substrate-related compounds, such as olive oil and oleic acid, the intracellular methanolysis activity strongly correlated with the relative amounts of the membrane-bound lipase, which suggests that ROL31 localized in the membrane plays a crucial role in the methanolysis activity of R. oryzae cells.     

Transesterification by lipase entrapped in electrospun poly(vinyl alcohol) fibers and its application to a flow-through reactor

We entrapped lipase in electrospun poly(vinyl alcohol) fibers of approximately 1 mum in diameter and evaluated the transesterification activity by converting (s)-glycidol to glycidyl n-butyrate with vinyl n-butyrate. The initial transesterification rate of the entrapped lipase was 5.2-fold faster than that of non-treated lipase. The fibrous membrane could be used as a component of a flow-through reactor for continuous transesterification.     

Partial purification and some properties of the lipase present in oil palm (Elaeis guineensis) mesocarp

The fatty layer obtained by centrifuging a homogenate of oil palm fruit mesocarp contains an active lipase. The lipase which was partially purified using a combination of ammonium sulphate fractionation, ion exchange and gel filtration chromatography, indicated an optimum activity at pH 4.5 and a temperature of 30°C. The enzyme exhibited good activity towards its natural substrate, palm oil as well as glycerol trioleate and glycerol tripalmitate. It also showed a linear reaction rate for the first 20 min of incubation. Sodium cyanide, resorcinol, cholesterol, lecithin and glycylglycine strongly inhibited its activity while phenol, L-cysteine and EDTA enhanced its activity. It is suggested that the lipase is associated with the membrane of the oil droplets.     

Lipoprotein lipase and lipolysis in milk

Bovine milk contains a lipoprotein lipase that accounts for most, if not all, of its lipolytic activity. The total lipase activity in raw milk is sufficient to cause rapid hydrolysis of a large proportion of the fat. However, in reality this does not happen, because the lipase is prevented from accessing the fat by the milkfat globule membrane. Physical damage to this membrane in raw milk initiates lipolysis. Furthermore, simply cooling certain individual milks soon after secretion can initiate the so-called spontaneous lipolysis. The biochemical basis of spontaneous lipolysis is still poorly understood, but it appears to be related to a balance between activating and inhibiting factors in the milk. Lipolysis in milk and milk products causes rancid off-flavours and other problems, and is a constant concern in the dairy industry. A thorough understanding of the mechanism of lipolysis and constant vigilance by operatives is required to minimize lipase-related problems.     

脂肪酶的膜固定化和酶膜反应器

详细讨论了脂肪酶的膜固定化方式和方法，综述了脂肪酶膜反应器的应用和动力学