反胶束体系中脂肪酶催化合成生物柴油

本文采用了实验室自制的Candida sp.99-125脂肪酶, 研究了其在丁二酸二酯磺酸钠(AOT)反胶束体系中, 催化大豆色拉油合成生物柴油的新方法。考察了溶剂极性、AOT浓度、W0(水与表面活性剂质量比)、缓冲溶液pH值、温度等因素对脂肪酶催化合成生物柴油的影响。研究结果表明: AOT/异辛烷反胶束体系为Candida sp.99-125脂肪酶催化提供了较为合适的微环境, 在W0为11, 表面活性剂浓度为50 mmol/L, 温度为40℃, 缓冲液pH值为7的AOT/异辛烷反胶束体系中, 醇油摩尔比为3∶1, 摇床转速为180 r/min, 采用12h3次流加1 mol当量的甲醇, 单批最高酯转化率可以达到90%。     

反胶团中脂肪酶催化性能的研究

研究了皱褶假丝酵母脂肪酶在AOT／异辛烷反胶团中催化橄榄油水解的性能。发现在25℃,pH7．G,R=[H₂O]／[AOT]：lO的条件下,脂肪酶的活力最高。腊肪酶在含水量较低的反胶团中较稳定,当R=5．4,于33℃保存12 h,活力可保持89％,底物浓度高达50％(v／V)时仍无抑制;当“水池”内存在40mmol／L的Gu²⁺、Co²⁺、Mn²⁺等金属离子时对酶仅有少许抑制。     

生姜蛋白酶提取及反胶束纯化工艺初步研究

本文研究了生姜中生姜蛋白酶的分布及贮藏中的活力变化 ,研究了从新鲜生姜中提取生姜粗蛋白酶及用AOT 异辛烷和CTAB庚烷 /辛醇反胶束萃取该酶的工艺和方法。实验结果指出 :在贮藏茎中生姜蛋白酶的活力为 2 .7μg/mL·min-1,在膨大茎中该酶活力为 0 .6 8μg/mL·min-1,而在幼嫩茎中活力最低 ,仅为0 .4 8μg/mL·min-1。新鲜生姜在 0℃下贮藏 2 4h即完全丧失活力 ,在室温下贮藏 3d后其活力损失达38 2 5 %。用 10倍 0 .2mol/L、pH =6 .0的磷酸缓冲液 (4℃ )三次提取生姜蛋白酶 ,其提取率分别为 6 4 .75 %、14 .2 8%和 5 .2 %。用 6 5 %饱和度的 (NH4) 2 SO4沉淀提取液中的生姜蛋白酶 ,再以 pH 6 .2、0 .1mol/L的柠檬酸缓冲液溶解 ,其比活力达到 4 .2 1(μgPro/ μgPro·min-1)。生姜蛋白酶的 pI =5 .4～ 5 .5 ,在pH 5 .4以上 ,用AOT 异辛烷反胶束不能萃取出生姜蛋白酶 ,但却可以萃取出 71.86 %的杂蛋白。用CTAB庚烷 /辛醇反胶束二次萃取AOT 异辛烷萃余液 ,其蛋白质萃取率为 6 0 .2 5 % ,萃取液中生姜蛋白酶理论比活力达到 4 9.77(μgPro/ μgPro·min-1)。     

复合诱变选育高产脂肪酶黑曲霉菌株及其固定化酶性质

通过硫酸二乙酯(DES)和微波复合诱变,获得遗传性状稳定的高产脂肪酶黑曲霉突变菌株CM2,酶活达174.93 U/mL.对菌株CM2培养条件的优化,以橄榄油和(NH_4)_2SO_4为最佳碳、氮源,在28℃、pH 7.5的条件下,发酵CM2菌株68 h,脂肪酶活为180.52 U/mL.大孔树脂固定化脂肪酶在35～55℃和pH 7.5～9.5之间有很好的稳定性.游离酶和固定化酶的表观失活活化能分别为52.6842 kJ/mol和30.8391 kJ/mol,固定化酶对温度的敏感度降低,耐受性增强.在微水相中脂肪酶催化2-辛醇和乙酸乙烯酯不对称酯交换反应中,(S)-乙酸辛酯的对映选择性高(游离酶e.e.s 85.7%;固定化酶e.e.s 87.7%),显示了该固定化酶在2-辛醇的手性拆分方面具有良好的应用前景.     

单宁酶反胶束微反应器生产没食子酸戊酯的研究

研究利用单宁酶微反应器生产没食子酸戊酯(amylgallate,AG)的方法。采用AOT(双(2乙基己基)磺基琥珀酸钠)异辛烷水组成的微反应器首次成功合成了没食子酸戊酯。并对反应体系中的各种主要参数对反应底物没食子酸(gallicacid,GA)的转化率的影响进行了探索。研究表明,反应条件为pH=6,温度45℃,[AOT]=020molL,振荡速度为150rmin时,W0=10或125(W0=[水][表面活性剂])的条件下,没食子酸的转化率在反应96h都可以达到90%。     

腐生葡萄球菌M36耐有机溶剂脂肪酶基因的克隆与原核表达

脂肪酶是重要的工业用酶,在食品加工、生物柴油的合成等领域具有广泛的应用。但是在应用中有机溶剂对脂肪酶具有一定的毒性,因此获得耐有机溶剂的脂肪酶基因并实现高效表达是脂肪酶规模化应用的前提。本研究应用PCR技术首次从耐有机溶剂脂肪酶产生菌腐生葡萄球菌M36基因组DNA中扩增得到脂肪酶Ⅲ基因lip3(GenBank AccessionNo.FJ979867),其编码区长度为741bp,编码247个氨基酸,推测蛋白分子量大小为31.6kD。它与腐生葡萄球菌lip3推测的基因(GenBank AccessionNo.AP008934)只有83%的同源性。将该基因与大肠杆菌表达载体pET-DsbA连接,转化大肠杆菌EscherichiacoliBL21(DE3)获得重组菌株BL21(DE3)/pET-DsbA-lip3,在pH8、25oC条件下,OD600为1.0时用0.4mmol/LIPTG诱导12h酶活达到25.8U/mL。重组酶在甲醇、正己烷、异辛烷、正庚烷等有机溶剂中具有较好的耐性。lip3基因的克隆及在大肠杆菌中有效表达的研究为进一步进行基因工程改造和脂肪酶应用奠定了基础。     

低温脂肪酶的产酶条件优化及其酶学性质

利用单因素筛选和正交试验对Burkholderia sp. SYBC LIP-Y发酵产酶的液体培养基和发酵条件进行了优化,其优化配方为：可溶性淀粉10 g/L、牛肉膏15 g/L、NaNO3 0.252 g/L、橄榄油40ml/L、Triton x-100 10ml/L、初始pH 7.5、接种量10%(V/V),脂肪酶酶活达到85.23U/ml,是优化前的3.63倍。通过对双水相纯化得到的脂肪酶进行酶学性质研究,确定该酶反应的最适pH为10.0,最适温度为30℃,40℃下保温60min酶活性还有80%以上,该脂肪酶为低温脂肪酶,热稳定性好,具有一定的耐醇性,应用前景广阔。     

非水介质中酶催化葡甘聚糖的转酯化反应

葡甘聚糖(KGM)是一种具有优异的生物降解性、生物相容性、独特的生理和药理功能的天然聚多糖。通过环境友好的、高选择性的酶催化转酯化反应引入疏水基团可以制备葡甘聚糖的酯化衍生物,从而拓宽其应用领域。本研究探讨了在非水介质中以脂肪酶Novozym 435、Lipozym RMIM、Lipozym TLIM和Lipase TypeⅦ催化KGM和乙酸乙烯酯的转酯化反应,并且考察了相关因素对转酯化反应的影响。实验结果表明:在非水介质N,N-二甲基乙酰胺、甲苯和异辛烷中,脂肪酶Novozym 435可以较好的催化乙酸乙烯酯和KGM发生转酯化反应。以Novozym 435为催化剂、以异辛烷为反应介质,当底物浓度[S]=30(mg/mL)、酶用量[E]/[S]=0.30(wt/wt)、酰基供体乙酸乙烯酯用量[Acyl]/[OH]=3.0(mol/mol)、50℃、反应72 h的条件下,酯化KGM的取代度(DS)可达到0.49。     

扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达

将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。     

响应面法快速优化曲霉Aspergillus sp.F044产脂肪酶培养条件

运用逐因子试验(Seriatim-Factorial Experiment)、Plackett-Burman、Response Surface Methodology(RSM)和单因子试验(Monofactorial Experiment)分析对产脂肪酶曲霉Aspergillus sp.F044产酶培养条件进行了快速优化。首先运用逐因子试验确定Aspergillus sp.F044产脂肪酶最适碳源和氮源,分别为麦芽糖和牛肉膏。在此基础上,通过Plackett-Burrman设计对影响其产酶相关因素进行评估并筛选出具有显著效应的橄榄油、牛肉膏、硫酸镁三个因素。然后通过实验拟合上述三因素与发酵液脂肪酶活力的一阶线性模型,沿着一阶模型给定的路径进行最速上升试验,在上升最高点处由中心组合试验和向应面分析确定其最优培养基组成;最后通过单因子试验确定最适发酵温度和摇床转速。试验优化的最优培养条件为:麦芽糖1.5%(w/v),硫酸铵7‰(w/v),磷酸氢二钾1‰(w/v),牛肉膏1.25%(w/v),硫酸镁2.11‰(w/v),橄榄油1.41%(v/v),自然pH,250r/min和30℃培养72h酶活达32.15U/mL。与初始11.18U相比,酶活提高2.88倍。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418