基于麦芽糖诱导型启动子在枯草芽胞杆菌中异源表达麦芽四糖淀粉酶及其酶学性质的研究

麦芽四糖淀粉酶(Maltotetraose amylase, Mta)可以从淀粉的非还原末端特异性依次切割第4个α-1,4糖苷键形成麦芽四糖,目前在食品、医疗保健和造纸等领域具有重要应用。构建安全、高效的表达系统强化麦芽四糖淀粉酶的重组表达,进而降低以其为核心酶的麦芽四糖生物转化过程的生产成本具有迫切的现实需求。本研究将源自Pseudomonas saccharophila(DSM 654)的麦芽四糖淀粉酶基因mta在枯草芽胞杆菌Bacillus subtilis中重组表达,利用麦芽糖诱导型启动子实现其安全高效表达,之后对重组酶进行分离纯化和酶学性质表征。结果显示,将携带麦芽糖诱导型启动子P_glv的表达载体转入B.subtilis WB800N中,成功构建工程菌后进行诱导表达,并且利用金属离子螯合层析技术成功获得了Mta纯酶。酶学性质研究结果显示其最适反应温度为55℃,最适反应pH为7.5。动力学常数K_m为(1.26±0.17) g/L、k_cat/K_m为(2 275.07±32.83) L/s·g,...     

芽四糖淀粉酶产生菌的筛选与发酵条件的研究

从290个土样中分离到1380株细菌,加上本所其他课题组提供的细菌共1870株,其中有707株能分解淀粉,经过复筛、纸层析鉴定有3株菌的淀粉酶酶解液中主要产物是麦芽四糖,进一步用β－淀粉酶水解为麦芽糖,用萄葡糖淀粉酶水解为萄葡糖,确证为麦芽四糖。其中最优菌株为537．1,其酶解产物中麦芽四糖占90％,而其他两株菌的酶解产物中除麦芽四糖外,还有较多的麦芽糖及麦芽三糖,因此选择了537．1作为形成麦芽四糖淀粉酶的优良菌株,经鉴定,该菌属于产碱菌(Alcaligenessp．)。菌株537．1产酶的较好条件为t培养基中麦芽糖1．5％,蛋白胨0．5％,起始pH7—7．5,在27—28℃振荡培养48h。株537．1培养液可以酶解谷类、薯类和野生植物淀粉生成麦芽四糖。     

产碱菌麦芽四糖淀粉酶的纯化及性质

产碱菌(Alcaligenes sp．)537．1除去菌体的培养液经硫酸铵沉淀及DEAE-纤维素离子交换柱层析,得到了凝胶电泳均一的麦芽四糖淀粉酶。纯化了141倍,酶活力回收40.1％,比活力达3308U／mg。用浓度梯度PAGE和SDS—PAGE测定酶分子量分别为68000和66000,不具亚基。用PAG-1EF测定等电点为4．45。酶反应最适pH和温度分别为7.0和60C。在pH7—10范围内稳定,该酶半衰期为37C 12小时,50 C 1小时和62 C 6分钟。 麦芽四糖淀粉酶是糖蛋白,含有4％左右的糖,含有27．10％酸性氨基酸、11.08％碱性氨基酸和1．82％色氨酸。     

海藻糖微生物酶法合成机制的研究

来源于嗜酸热古菌芝田硫化叶菌(Sulfolobus shibatae)B12的麦芽寡糖基海藻糖合酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)基因在大肠杆菌中获得表达。将获得纯化的两个酶,分别以麦芽寡糖和淀粉为转化底物,在pH5.5,60℃条件下合成海藻糖。从反应产物分析结果可知,两个酶合成海藻糖时能利用的最小底物是麦芽四糖,海藻糖产率与麦芽寡糖链长正相关。同时还发现两个酶都具有轻微的α-1,4-葡萄糖苷酶活性,能在麦芽寡糖还原末端水解α-1,4糖苷键,生成葡萄糖分子,其反应最小底物分别是麦芽三糖和四糖。推测海藻糖合成酶可能有两个不同的催化活性中心。     

产碱菌麦芽四糖淀粉酶水解淀粉的特性

产碱菌麦芽四糖淀粉酶水解不同来源淀粉的产物组成有差异:G4占81.5％～98.8％,G_3占0％～9.6％,G_2占0％～5.9％。不同淀粉的水解速度在4170～9036mgGlch~-(1)·mg~(-1)之间。对可溶性淀粉的水解产物为6-型。麦芽四糖淀粉酶能被小麦、玉米及马铃薯的生淀粉吸附,其吸附率分别为60.2％、50.0％及52.2％,相对水解率分别为4.5％、2.7％及0％,水解生淀粉的主要产物为G_4。     

链霉菌Streptom yces olivaceusFXJ7.023多功能葡糖淀粉酶SoGA的克隆、表达及鉴定

根据海洋来源链霉菌Streptomyces olivaceus FXJ7.023基因组测序结果设计特异引物,通过PCR扩增获得1条全长为1 788 bp的糖苷水解酶15家族蛋白新成员完全编码区DNA片段,该片段编码1个595个氨基酸残基、分子量为66.2 k D的预测蛋白。利用基因工程技术将该片段重组入原核表达质粒p ET32a并转化宿主菌BL21(DE3)ply Ss,IPTG诱导融合蛋白表达,表达的包涵体融合蛋白经纯化、复性后利用DNS法测定其在不同温度、p H条件下催化不同底物产生还原糖的活性。结果表明,该酶能够水解纤维素、淀粉等多种底物产生还原糖活性,且对不同底物表现不同的最适p H和最适反应温度。     

Thermococcus sp.B1001环糊精酶的重组表达及在麦芽七糖制备中的应用

成功构建pET-24a-tscd质粒,实现Thermococcus sp.Strain B1001来源的环糊精酶(TsCDase)在Es-cherichia coli BL21(DE3)中表达.通过热处理和镍柱分离对重组TsCDase进行纯化.酶学性质研究表明,重组TsCDase的比活为1 208.04 U/mg,最适温度为90℃、最适pH值为5.5.重组酶TsCDase在85℃、90℃、95℃条件下的半衰期分别为180、120、30min.酶转化研究表明,以80 g/L β-环糊精为底物,当酶转化温度为90℃、反应pH值为5.5～6.0,加酶量为25 U/g,反应时间为4 h时,麦芽七糖产率为81.19％和85.95％,七糖占产物麦芽寡糖的比例为95.24％和92.92％.本研究结果为工业化制备麦芽七糖奠定良好基础.     

Thermococcus sp.B1001环糊精酶的重组表达及在麦芽七糖制备中的应用

成功构建pET-24a-tscd质粒,实现Thermococcus sp.Strain B1001来源的环糊精酶(TsCDase)在Es-cherichia coli BL21(DE3)中表达.通过热处理和镍柱分离对重组TsCDase进行纯化.酶学性质研究表明,重组TsCDase的比活为1 208.04 U/mg,最适温度为90℃、最适pH值为5.5.重组酶TsCDase在85℃、90℃、95℃条件下的半衰期分别为180、120、30min.酶转化研究表明,以80 g/L β-环糊精为底物,当酶转化温度为90℃、反应pH值为5.5～6.0,加酶量为25 U/g,反应时间为4 h时,麦芽七糖产率为81.19％和85.95％,七糖占产物麦芽寡糖的比例为95.24％和92.92％.本研究结果为工业化制备麦芽七糖奠定良好基础.     

嗜碱菌碱性淀粉酶的研究

分离自内蒙古自治区察汗淖碱湖的嗜碱菌株No．1 0-1,好气,运动,细胞杆状,革兰氏染色阴性。该菌生长pH范围为8．0—13．0,最适生长pH1 0.0-ll.0,为专性嗜碱菌。在含淀粉培养基中产生胞外碱性淀粉酶,最适产酶条件是： 碳源为土豆淀粉,氮源为复合蛋白胨,Nacl浓度为2．O％,Na2CO3浓度为1．0—1．5％(pH9．9-10．5)。 酶的最适反应pH为10．0,稳定pH8．0,最适反应温度为50℃。作用于直链淀粉其水解产物为β-构型,主要产物是麦芽糖,其次为麦芽三糖、葡萄糖和麦芽四糖。嗜碱菌No.10-1产生的酶为碱性β-淀粉酶。     

一种新型淀粉酶的鉴定及其产酶菌株的筛选*

对筛选到的菌株ZX99产生的一种新型淀粉酶 (异麦芽低聚糖酶 )进行了分析鉴定。ZX99菌株能产生一种胞外淀粉酶 ,该酶能催化淀粉的降解产生异麦芽低聚糖。对原产酶菌株ZX99多次进行紫外线照射诱变后 ,获得了优良、稳定的变异菌株BS3.232 ,其产酶水平为原株的160 %。产物薄层层析证明 ,该酶能催化淀粉的降解 ,产生异麦芽糖、潘糖、异麦芽三糖和异麦芽四糖等低聚糖 ,但对普鲁兰基本不起作用 ,由此证明它是一种不同于新型普鲁兰酶 (neopullulanase)和传统淀粉酶 (amylase)的一种新型     

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。     

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.     

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.     

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.     

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。