超临界CO2萃取红景天中红景天苷、苷元酪醇的研究

采用超临界CO₂ 萃取法和乙醇常温浸提法相比较, 研究从红景天中提取红景天苷、苷元酪醇的工艺条件, 结论是:采用超临界CO₂ 萃取法能萃取出红景天生药中红景天苷的1.2%, 提取率不高, 但该方法能萃取出80%的苷元酪醇, 萃取液中苷元酪醇的相对含量可达45.68%;乙醇常温浸提法能将红景天苷、苷元酪醇同时有效萃取, 且得率较高, 但是萃取液中两物质相对含量较低, 进一步分离纯化将有难度。本研究结果表明, 将超临界CO₂ 萃取法和乙醇常温浸提法有效结合, 可实现两物质的有效分离, 推进红景天有效成分的产业化进程。     

微生物法合成红景天苷

红景天苷是红景天属植物的主要有效成分之一,具有耐缺氧、抗辐射、抗疲劳、抗肿瘤、降血糖、提高免疫力等多重功效。随着其需求量的日益增加和植物资源的不断减少,微生物法合成红景天苷因具有周期短和易调控等优势而倍受关注。目前微生物法合成红景天苷尚处于基础研发阶段,为了方便相关领域研究者系统了解其研究现状和探讨其未来发展方向,文中对红景天苷生物合成途径、糖基转移酶、野生菌/天然酶资源和工程菌/重组酶体系进行了综述。     

不同生境鱼腥草形态特征及主要有效成分含量差异分析

以不同生境中的鱼腥草为研究对象,分析其形态特征及主要有效成分含量的差异。该文测定了不同生境下主要环境因子指标及鱼腥草的叶长、叶宽、株高、基径、生物量和含水量,用高效液相色谱法测定其各部分主要有效成分金丝桃苷、槲皮苷含量,并对测量结果进行单因素方差分析和相关性分析。结果表明:环境因子会对鱼腥草的形态建成及有效成分积累造成不同影响,适宜的光照强度有利于鱼腥草对有效成分的累积;鱼腥草中有效成分的含量表现出叶茎根的规律。适宜的遮阴处理,既可避免强光照对鱼腥草生长的抑制,又可显著提高有效成分的含量,从而整体增加对鱼腥草有效成分的收益。     

红景天苷对运动后自由基和能量代谢改变的影响

目的:探讨红景天苷在运动过程中对自由基和能量代谢相关指标的作用,从抗氧化系统、能量代谢系统等方面研究红景天苷抗运动性疲劳的机制。方法:本研究采用小鼠运动模型,40只雄性小鼠随机取分为4组(n=10):红景天苷运动组,红景天苷安静组,运动对照组,安静对照组。红景天苷运动组和安静组两组以150 mg/(kg.d)的红景天苷灌胃给药,运动和安静对照组两组以同样体积蒸馏水灌胃,连续给药2周;末次灌胃30 min后,运动组进行无负重游泳120 min,然后测定与运动性疲劳相关的生化指标。结果:研究结果表明,红景天苷能够提高运动小鼠肝脏内超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶活性,降低丙二醛(MDA)含量;红景天苷具有稳定运动小鼠血糖,增加肝、肌糖原储备,防止长时间运动后血糖和肝、肌糖原水平降低的作用;红景天苷可提高运动小鼠血浆总胆固醇(TC)、甘油三酯(TG)及游离脂肪酸(FFA)的水平,有影响不同状态下的脂肪代谢,促进脂肪利用的作用。结论:红景天苷对运动过程中自由基和能量代谢改变的影响是其抗运动性疲劳的机制之一。     

两种耐热磷酸化酶的构建及高效催化合成红景天苷

目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。     

海藻预处理工艺研究

大型海藻是中含有大量的糖分,能被微生物利用进行发酵作用来生产乙醇。为了提高乙醇的产率,优化预处理方法是必要的。本课题主要通过物理研磨法、蒸煮法、酸解法和酶解法处理海藻以优化出糖类溶出量最大的预处理条件。物理研磨法条件为固液比1:20,温度28℃,处理2h;蒸煮法条件为固液比1:30,温度90℃,处理1.5h;酸解法条件为固液比1:30,温度为80℃,处理1h;酶解法条件为酸解液残渣在温度40℃下酶解3h。且酸解后进行酶解的处理方法是糖类溶出量最大的处理方法,条件为80℃的酸解残渣在纤维素酶的作用下40℃酶解3h。     

用纯制人血纤维蛋白溶酶脱除人免疫血清球蛋白中的抗补体活性

<正> 用高纯度的人血纤维蛋白溶酶处理人免疫血清球蛋白制剂,以脱除其抗补体活性。从免疫血清球蛋白制剂中去除抗补体活性的程度和所获得的分子分裂量取决于加入的血纤维蛋白溶酶量及其作用的时间。血纤维蛋白溶酶反应作用则通过加入皂土吸附、去除使之仃止。获得了两种类型的可用于静脉输注的、血纤维蛋白溶酶处理的免疫血清球蛋白。一种为较多裂解产物(35-45％)较低抗补体活性,另一种为较少裂解产物(15-20％)、含较高抗补体活性。前者的抗体稍被减少而后者基本上为未改变的、未经处理的原血清免疫球蛋白。     

通过定点突变提高纳豆激酶纤溶活性和热稳定性

纳豆激酶（Nattokinase, NK）是一种纤溶酶，可溶解血栓，常用于治疗心血管疾病（CVDs）。然而，野生型NK往往表现出很少的纤溶能力和较低的热稳定性，针对如何提高NK的热稳定性和活性开展研究。使用重叠延伸PCR法将NK中位于194位的Ser替换为Pro，为验证突变后的热稳定性，将野生型NK和突变体S194P均在不同温度下孵育相同时间，使用纤维蛋白板法测定二者酶活，验证热稳定性。成功构建了pET-26b-NK_S194P，测量酶活结果显示，野生型NK和突变体S194P酶活分别达到101.30 IU/mL和123.23 IU/mL，结果表明突变体S194P的酶活比野生型NK高（18.10±2）%，将野生型NK和突变体S194P在60~65 ℃温度下孵育30 min后测量酶活，野生型NK在63 ℃时丧失酶活，突变体S194P在65 ℃时丧失酶活，耐热温度提高2 ℃。结果表明，突变体S194p的蛋白结构在突变后发生了改变，使NK提高了耐热温度。     

红景天苷生物、化学和生物催化合成的分子理论及应用

红景天苷是红景天属植物的主要活性成分之一,研究红景天苷的高效合成具有重要的科研和应用价值。本文全面概述近年来合成红景天苷的研究状况,主要包括生物合成途径、化学合成途径、生物催化合成途径。对生物合成途径中红景天苷的代谢合成途径、关键酶及基因、实践现状进行了分析;总结了以Koenigs-Knorr法为理论基础的化学合成的研究进展;对具有广泛发展前景的体外生物催化合成状况进行了理论与实践的概述。通过对这些合成方法的展望,为人们了解合成红景天苷的研究现状、进一步深入研究提供参考。     

ATP对菠菜二磷酸核酮糖羧化酶的热稳定作用

纯化的菠菜RuBP羧化酶有热易变性。加入ATP对RuBP羧化酶的热失活有明显的保护作用。如同时加入DTT则可进一步提高ATP对酶的热稳定作用。此外,ADP,AMP和无机磷也有热稳定作用,但效果比ATP差。照光叶子的ATP含量比暗处理的叶子高一倍,而其RuBP羧化酶粗提液在60℃保温后的酶蛋白含量及酶活力分别比暗处理的高3至7倍。如果在暗处理的RuBP羧化酶粗提液中加入外源ATP时,则酶的热稳定性可达到光处理的86％。     

Ease of solubilization of five marker enzymes in three preparations of rat renal brush border membranes

The ability of eight stripping agents to solubilize five marker enzymes from rat renal brush border membranes isolated by three different preparative methods was examined. Protein and enzyme activities - alkaline phosphatase (APase), L-leucine aminopeptidase (LAPase), gamma-glutamyl transpeptidase (GGTase), gamma-glutamyl hydrolase (GGHase) and maltase - solubilized by the treatments were expressed as percent of total activity recovered in excess of control values. The relative enzyme activity and the solubilization factor were determined for each marker enzyme in every treated sample and the treatments with the eight agents compared. Trypsin treatment released > 80% of LAPase and < 10% of total membrane protein. Papain treatment released only 16--23% of total membrane protein but most of the enzyme activities except APase. Neuraminidase had no solubilizing effect. 4--10% of total membrane protein was solubilized by LiCl treatment but no marker enzyme activities were released. Less total membrane protein was released by treatment with proteolytic enzymes or LiCl than with the detergents Triton X-100, hexadecyltrimethylammonium bromide, sodium deoxycholate, and sodium dodecylsulfate. APase activity was the least readily solubilized. Correlating the degree of solubilization for five marker enzymes with the types of stripping agents used and with the appearance of the membrane surface when examined by electron microscopy led to the suggestion that LAPase, GGTase, GGHase and maltase molecules are part of an interwoven surface layer of membrane proteins which can be disrupted by transamidation and transesterification reactions. APase appears to be more strongly associated with the intact lipid matrix than the bulk of the membrane protein.     

Solid-state ethanol fermentation by means of inert gas circulation

A new method for solid-state ethanol fermentation (the SSEF system) was experimented on for the ethanol production from solid starchy materials, where a packedbed-type fermentor was used. Both cultivation of Aspergillus saitoi and enrichment of a saccharifying enzyme were effective for hydrolysis of the starch. Ethanol production was set in by a form of parallel fermentation using a respiration-deficient mutant of Saccharomyces cerevisiae. Produced ethanol was simultaneously stripped by circulating inert gas and separated in a condenser. Average ethanol concentration in the condensate was over 200 g/L, and over 90% of produced ethanol was recovered from the packed bed during 15 or 16 days of stripping. The fermentation efficiency was about 80%, which was evaluated much higher than those of conventional solid-state fermentations. The residue had lesser volume and a higher solids content compared with the distillery wastewaters of conventional liquid-state fermentations. This means an advantage for the treatment and the effective conversion of the residue into fetilizers or animal feeds.     

利用甲基对硫磷降解菌DLL-E4消除农产品表面农药污染的研究

利用甲基对硫磷降解菌DLL-E4(Pseudomonas sp.)能高效分解甲基对硫磷的特性,研究了消除农产品表面农药残留污染的途径,采用菌体发酵液、发酵液上清液、菌体蛋白、粗酶液、添加菌体蛋白的洗涤剂以及洗涤剂,处理了小青菜、茶叶、黄瓜表面的农药残留污染。结果表明,使用发酵液和不同的酶制剂可以有效去除这些产品表面的农药残留污染,最高去除率可达100％;DLL-E4上清液中有一些酶促反应促进因子,有助于酶更好地发挥作用,洗涤剂和酶直接混合作用,有助于消除茶叶表面的农药残留,但对于黄瓜和小青菜则相反。在所有选用的模式中,使用粗酶液和菌体蛋白效果最好,对黄瓜、小青菜和茶叶合适的酶制剂浓度分别为2％、5％和10％,粗酶液同时还能降解甲胺磷、辛硫磷、毒死蜱等农药。     

酶法提取铁皮石斛多糖工艺优化及对挥发性成分的影响研究

铁皮石斛具有重要的药用价值,但其资源有限,因此对其充分开发和利用具有重要的意义。本文利用α-L-鼠李糖苷酶辅助提取铁皮石斛多糖,通过对酶解温度、酶解时间及加酶量三个因素进行优化,提高铁皮石斛多糖提取率;结果显示,在酶解温度为40℃、酶解时间为1 h、加酶量为2.5%时,多糖提取率为38.4%,而不加酶处理多糖提取率仅为21.7%,酶法处理大大增加了铁皮石斛的多糖提取率。同时,运用顶空固相微萃取技术结合气相色谱质谱联用仪(HS-SPME-GC-MS)分析比较α-L-鼠李糖苷酶对铁皮石斛挥发性成分的影响;GC-MS分析表明,铁皮石斛加酶处理前后共发现31种挥发性成分,主要成分均为己醛、1-辛烯-3-醇、异薄荷醇和3-辛烯-2-酮,并且醛类化合物在加酶处理前后的样品中相对含量均最高,且加酶处理后产生6种新的挥发性成分。该研究结果表明α-L-鼠李糖苷酶的使用可显著增加对铁皮石斛多糖提取率,并为铁皮石斛挥发性成分的研究和开发利用提供一定的参考依据。     

A microreactor for the study of biotransformations by a cross-linked γ-lactamase enzyme

A (+)-γ-lactamase was precipitated, cross-linked and the resulting solid crushed prior to immobilisation within a capillary column microreactor. The microreactor was subsequently used to study enzyme stability, activity, kinetics and substrate specificity. The thermophilic (+)-γ-lactamase retained 100% of its initial activity at the assay temperature, 80°C, for 6 h and retained 52% activity after 10 h, indicating the advantage of immobilisation. This high stability of the immobilised enzyme provided the advantage that it could be utilised to screen many compounds in the microreactor system. This advantage overcame the fact that the immobilisation process affected enzyme kinetics and activity, which was reduced (by 70%) compared to the free enzyme. In general, the enzyme displayed similar substrate specificity to that found in a previous study for the free enzyme; however, enhanced activity was seen towards one substrate, acrylamide. The system developed correlates well with the free enzyme in batch assay and indicates the suitability of the system for enzyme substrate screening, allowing a significant reduction in cost, due to the reduced amounts of enzyme, substrates and other assay constituents required.     

Evidence for the Functional Association of Enzyme I and HPr of the Phosphoenolpyruvate-Sugar Phosphotransferase System With the Membrane in Sealed Vesicles of Escherichia coli

Several independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate-dependent sugar phosphorylation was catalyzed by this membrane-bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS-enzyme complex was stabilized by Mg⁺⁺ and phosphoenolpyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.     

卵巢囊肿剥除术对卵巢囊肿患者术后卵巢储备功能的影响

目的:探究卵巢囊肿剥除术对卵巢囊肿患者术后卵巢储备功能的影响。方法:选取我院收治的卵巢囊肿患者42例,按照随机数字表法分为实验组和对照组。对照组20例,采用经腹卵巢囊肿剥除术;实验组22例,采用经腹腔镜下卵巢囊肿剥除术。对比两组术后6个月血清中的抗苗勒管激素(AMH)、卵泡刺激素(FSH)及雌二醇(E2)水平,超声下卵巢体积(Vol),窦状卵泡数(Fo)水平等。结果:治疗后,实验组总有效率为(95.5%)高于对照组总有效率(75.0%),差异有统计学意义(P0.05);与治疗前相比,两组患者术后第6个月血清FSH、E2水平及Vol、Fo水平无明显变化,差异无统计学意义(P0.05);与治疗前相比,两组患者血清AMH水平较低,差异有统计学意义(P0.05),且组间比较差异无统计学意义(P0.05)。与对照组相比,实验组术中出血量较低,手术时间较少,下床活动时间短(P0.05)。结论:卵巢囊肿剥除术对于卵巢储备功能的恢复具有积极作用,有利于保证育龄女性卵巢功能的完整,促进各基础性激素水平的恢复,尤其是采用腹腔镜卵巢囊肿剥除术出血少损伤小,病变清除彻底,术后恢复好。     

Evidence for active synthesis of two allelic b locus markers by single lymphocytes from heterozygous rabbits.

The double expression on single cells of both b4 and b6 allotypic markers by the mixed antiglobulin technique was observed on single peripheral blood lymphocytes (PBL) from heterozygous rabbits which were treated with pronase and incubated overnight to allow for regrowth of surface immunoglobulin (Ig). The percent of double staining rosettes observed after lymphocyte processing prior to pronase stripping ranged from 10–30%. Following pronase treatment at concentrations allowing for maximum stripping but assuring high cellular viability, the total rosetting population was reduced to 1–3% in the majority of cases. Occasionally, pronase stripping was less effective. Overnight incubation in serum free medium resulted in the reappearance of surface Ig with double staining rosettes ranging from 8–30%. Exposure of b6 or b4 normal rabbit serum to b4 or b6 cells, respectively, resulted in uptake of b6 in which most of the passively acquired Ig eluted off during overnight incubation, while negligible uptake of b4 was seen. Coincubation of normal b4, 4 PBL which presumably are capable of donating Ig with pronase treated b6, 6 PBL and vice versa also resulted in the absence of mixed rosettes. That simultaneous active synthesis of both allelic markers results in double allotype expression was demonstrated by kinetics data. Supporting this was the observation that only after washing of cycloheximide inhibited cells did allotype reexpression resume.     

Enhanced decolourization of Direct Red-80 dye by the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> employing sequential design of experiments

Decolourization of Direct Red 80 (DR-80) by the white rot fungus Phanerochaete chrysosporium MTCC 787 was investigated employing sequential design of experiments. Media components for growing the white rot fungus were first screened using Plackett-Burman design and then optimized using response surface methodology (RSM), which resulted in enhancement in the efficiency of dye removal by the fungus. For determining the effect of media constituents on the dye removal, both percent dye decolourization and specific dye removal due to maximum enzyme activity were chosen as the responses from the experiments, and the media constituents glucose, veratryl alcohol, KH₂PO₄, CaCl₂ and MgSO₄ were screened to be the most effective with P values less than 0.05. Central composite design (CCD) followed by RSM in the optimization study revealed the following optimum combinations of the screened media constituents: glucose, 11.9 g l⁻¹; veratryl alcohol, 12.03 mM; KH₂PO₄, 23.08 g l⁻¹; CaCl₂, 2.4 g l⁻¹; MgSO₄, 10.47 g l⁻¹. At the optimum settings of the media constituents, complete dye decolourization (100% removal efficiency) and a maximum specific dye removal due to lignin peroxidase enzyme of 0.24 mg U⁻¹ by the white rot fungus were observed.     

Potential allelochemicals from the essential oil of Ruta graveolens

The essential oil of aerial parts of Ruta graveolens was obtained by hydrodistillation with a 0.74% yield on a dry weight basis. Thirty-eight components were identified by GC and GC-MS analyses. 2-Ketones predominated in the essential oil, with undecan-2-one (46.8%) and nonan-2-one (18.8%) as the main constituents. The essential oil and some of its constituents were tested for their allelopathic activity in vitro on radish germination and radicle growth in light and darkness. The essential oil and some of its minor constituents were effective and dose-dependent inhibitors of both the germination and radicle growth; 2-ketones are not active. The possible allelopathic activity of rue essential oil and some its isolated constituents is reported.