利用内源元件在谷氨酸棒杆菌中分泌表达木聚糖酶

以来自于谷氨酸棒杆菌内源AH6启动子和5′UTR及其前38 bp结合合适的Shine-Dalgarno (SD)序列,构建双顺反子表达载体对木聚糖酶进行表达。为了能够实现分泌表达,选取了来自谷氨酸棒杆菌的两种分泌途径的信号肽,分别为Tat型的CgR0949及Sec型的CspB信号肽。在实现分泌表达之后,对其进行5 L发酵罐的扩大培养以提高分泌量。并对纯化的木聚糖酶进行了部分酶学性质的研究,包括最适催化pH及酸碱耐受性;最适催化温度及热稳定性。结果表明:在上述表达体系中,以CgR0949为信号肽木聚糖酶不能分泌到胞外;木聚糖酶能在CspB信号肽的引导下分泌到胞外,分泌表达量为486.2 U/mL。木聚糖酶的分泌量在5 L发酵罐水平上达到1 648.7 U/mL,是摇瓶培养的3.4倍。该木聚糖酶的最适反应pH为4.5,最适温度为45℃;在pH 4–11范围内4℃处理24 h酶活保持在80%以上;在50℃前处理15 min酶活保持在95%以上,超过60℃则酶活迅速下降至20%及其以下。上述结果表明,谷氨酸棒杆菌内源元件能有效用于木聚糖酶的分泌表达,扩大培养能进一步提升木聚糖酶的分泌量。该双顺反子表达体系能为外源蛋白在谷氨酸棒杆菌中的分泌表达提供一种可用的工具。此外,通过酶学性质的研究可进一步提高木聚糖酶的催化效率。     

信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响

枯草芽孢杆菌具有良好的安全性和优异的外分泌能力,是目前常用的脂肪酶异源分泌表达体系。在枯草体系中如何提高脂肪酶分泌量是目前的研究热点。该体系中信号肽与目的基因不匹配和目标蛋白总表达量不高是导致脂肪酶LipS分泌表达效果较差的重要原因。针对这些问题,依据信号肽结构特征的不同(正电荷与疏水氨基酸个数的差异)对枯草芽孢杆菌体系中Sec和Tat这两个主要分泌途径的信号肽进行了筛选,结果显示Tat途径中的phoD信号肽分泌效果明显优于其他信号肽。在此基础上,通过替换使用诱导型表达载体、突变优化phoD信号肽,并考察了表达条件的影响,显著提高了脂肪酶LipS的分泌表达量,发酵液中转酯酶活达到62.07U/L,占总酶活的62.30%,较原始分泌表达体系的分泌量提高了13.7倍。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

嗜热子囊菌JCM12803的α-半乳糖苷酶基因tcgal27A在毕赤酵母中的表达

从嗜热子囊菌(Thermoascus crustaceus)JCM12803克隆α-半乳糖苷酶基因,并对其酶学性质进行系统的研究,旨为获得工业应用性质优良的α-半乳糖苷酶。通过RT-PCR方法从T.crustaceus JCM12803中克隆α-半乳糖苷酶基因序列,并对其酶学性质进行了系统的分析。结果显示,tcgal27A属于糖苷水解酶27家族,基因全长1 918 bp,含有4个内含子,c DNA全长1 419 bp,编码472个氨基酸,预测tcgal27A N端含有24个氨基酸为其可能的信号肽。将TCGal27A在毕赤酵母中成功地进行了表达,所获得的重组TCGal27A具有高比活(336.5 U/mg)及宽泛的底物特异性,对蜜二糖(14.4 U/mg)、棉子糖(9.1 U/mg)、角豆胶(3.6 U/mg)、魔芋粉(1.6 U/mg)、瓜尔豆胶(1.3 U/mg)、水苏糖(0.7 U/mg)都有着不同程度的降解作用。以pNPG为底物时的Km值为1.6mol/m L,Vmax为536.8μmo L/(min·mg)。TCGal27A的最适作用pH为4.5,最适温度为65℃,50℃处理1 h之后酶活力还能保持86.0%。这些结果表明耐高温TCGal27A性质优良,可添加到饲料中,在消化和提高豆粕蛋白能量利用率方面有良好的潜在应用价值。     

N-氨甲酰水解酶的纯化及性质

从Burkholderiacepecianjut1分离纯化N氨甲酰D氨基酸水解酶(NDase)。实验表明,该酶亚基35KD,最适温度为52℃,最适pH为7.2左右。以N氨甲酰D苯丙氨酸作底物,其米氏常数Km为10.22mmol/L,最大反应速度Vmax为0.27mmol/(L·min)。实验表明二价金属离子对酶活有重要影响。     

胸腺腺苷脱氨酶免疫亲和纯化和性质研究

用免疫亲和层析结合常规生化方法从人胸腺中纯化腺苷脱氨酶达电泳纯, 比活力14898U/mg, 得率34.15%.该酶分子质量为41.3ku, 约由380个氨基酸残基构成, 等电点为4.9, 最适温度37～40℃, 最适pH为7.0以腺苷或2-脱氧腺苷为底物, 其K_m分别为83μmol/L和61μmol/L. 对氯汞苯甲酸能显著抑制酶活性, 二硫苏糖醇可使被抑制的活性得到部分恢复.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。     

来源于耐碱真菌Pseudallescheria sp. JSM-2的碱性木聚糖酶基因的克隆、表达及其性质研究

从造纸废水中分离得到的耐碱真菌Pseudallescheria sp. JSM-2的DNA为模板,利用同源克隆和TAIL-PCR的方法,获得了一个碱性木聚糖酶基因xyl11-1。该基因DNA和cDNA分别为797 bp和678 bp。该基因的推测蛋白N-端有一个18个氨基酸的信号肽序列和一个含207个氨基酸的成熟蛋白。编码成熟蛋白的cDNA序列在毕赤酵母GS115中重组表达后,进一步纯化并进行酶学性质测定。重组XYL11-1的最适pH为6.5,在pH 4.5~9.0范围有50%以上的酶活;在pH 4.5~12.0范围具有良好的pH稳定性;最适温度为50℃;以燕麦木聚糖为底物,比活为2 618 U/mg;且对中性和碱性蛋白酶具有极好的抗性。该酶作用底物范围广,包括各种木聚糖、纤维素和葡聚糖,易于工业化发酵生产,具有在纸浆脱墨、动物饲料、鱼类饵料中的应用潜力。     

白地霉Y162脂肪酶基因克隆及其在毕赤酵母中的高效表达

借助生物信息学,对已克隆的地霉属脂肪酶全长基因序列进行同源比对,根据保守序列设计引物,在基因组DNA和cDNA水平上,于国内首次克隆了Geotrichum candidum Y162脂肪酶基因.Gcandidum Y162脂肪酶基因全长1692bp,不含内含子,编码包括19个氨基酸信号肽在内的563个氨基酸.与NCBI GenBank中已报道的地霉属脂肪酶氨基酸序列有86%的一致性.将该基因克隆到pPIC9K表达载体上,转化毕赤酵母GS115,摇瓶发酵96h后毕赤酵母分泌表达55 U/mL重组脂肪酶,实现了脂肪酶的高效表达.酶学性质研究表明,该重组脂肪酶对C9位顺式双键的甘油酯具有明显的底物特异性;对甲醇、甘油等有机溶剂呈现耐受性;最适温度和最适pH分别为50℃和8.0,在pH6.0～10.0及60℃以下能保持60%以上的酶活力.底物特异性、有机溶剂、温度及pH耐受性赋予该重组酶良好工业应用潜力.     

重组a-氨基酸酯水解酶合成头孢曲嗪

为了探索酶法合成头孢曲嗪的产业化工艺路线,从红纹黄单胞菌Xanthomonas rubrillineans中克隆-氨基酸酯水解酶基因全序列,转化入大肠杆菌中表达。以头孢曲嗪的合成转化率为指标,分别考察纯化的重组-氨基酸酯水解酶合成头孢曲嗪的最适温度、最适pH和最佳底物摩尔比。经聚丙烯酰胺凝胶电泳分析,重组-氨基酸酯水解酶的单体分子量为70 kDa。催化合成头孢曲嗪的最适pH为(6.0±0.1),最适温度为36℃。底物浓度约为7-ATTC 30 mmol/L、HPGM HCl 120 mmol/L,酶用量22 U/mL时,头孢曲嗪的转化率达到64.3%。结果为优化酶法合成头孢曲嗪的产业化工艺奠定了基础。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.