部分酶解酵母高效电击转化研究

以酵母质粒YCp50为外源DNA,电击转化部分酶解酵母宿主菌AB1380,转化效率稳定在10~6转化子/μg质粒DNA左右,比不酶解酵母或酵母原生质球作受体的电击转化效率高一个数量级以上,也比PEG介导的酵母原生质球转化高3～5倍,而且适合于大片段DNA如水稻YAC分子的转化。达最佳转化时的有关技术参数为:新接菌种通气培养至细胞密度1×10~8～1.5×10~8个/ml;转化时细胞密度控制在1×10~9～1.5×10~9个/ml;每毫升酶解缓冲液加15u溶菌酶(lyticase),30℃下处理酵母5min进行部分酶解;电击时,电场设置在6.25kV/cm、电容25μF,电击后直接铺板。     

对酶概念的挑战

概述了Ｒｉｂｏｚｙｍｅ、抗体酶、生物酶工程生产的酶、人工酶、模拟酶、博莱霉素等最新科研成果,对传统的酶概念提出了严峻的挑战,并提出生物催化剂与化学催化剂两概念之间的鸿沟必将被填平。     

邻单胞菌(Plesiomonas sp.90-1)降解直链烷基苯磺酸钠酶的诱导

在Plesiomonas sp.90-1中,降解直链烷基苯环酸钠(LAS)相关的酶为诱导酶.使用正交多因子法研究了细胞降解LAS酶活诱导的最佳条件为:细胞培养温度30℃,LAS诱导浓度10ppm,酵母膏0.008%,pH8.0,通气.在此条件下,LAS酶活比未经诱导者提高1.4倍.碳源的加入会阻遏LAS降解酶的活力形成.在所试氮源中,以(NH_4)H_2PO_4对LAS降解酶的形成最有利.低浓度的磷酸盐对LAS降解酶活形成无影响,但高浓度的磷酸盐对LAS降解酶活形成不利.利用微量检压技术发现经此条件诱导的细胞耗氧量上升2—3倍.     

氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去污剂十二烷基硫酸溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化,而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度。在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。     

反应介质对脂肪酶稳定性的影响

脂肪酶Ｌｉｐｏｚｙｍｅ＾ＩＭ在有机溶剂中的热稳定性显著提高,在水溶液中,热处理温度高于５０℃后,其催化甘油三酯水解的活力迅速下降,温度升到６０℃时,水解活力仅残存１３．６％,温度高于７０℃该酶完全失活,而在有机溶剂正庚烷中,温度高达８５℃仍表现出较高的活性,反应介质的疏水性越强,酶抗超声辐射变性作用的能力越强,经同样的超声辐射处理后,正已烷中Ｌｉｐｏｚｙｍｅ＾ＩＭ的酯水解活力仅损失１１．８％,而在     

热锻炼能提高菜豆细胞可溶性蛋白,SOD,膜ATP酶和质子泵在体外的…

以菜豆为实验材料,研究了蛋白质分子和生物膜在体外热稳定性的变化和原因,实验结果表明：热锻炼可增加细胞的耐热性,并赋予ＳＯＤ、膜ＡＴＰ酶和生物膜质子在体外的热稳定性;通过对可溶性蛋白热变性后的浊度分析和用ＡＮＳ荧光探针探测蛋白质的结构变化,发现蛋白热变性的原因是疏水区的暴露和相互轩聚,＾３５Ｓ－Ｍｅｔ标记分析和二维电泳谱揭示,热锻炼诱导合成的热激蛋白具有阻止蛋白质热变性和生物膜热破坏的功能。     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

用平板透明圈法测定延胡索酸酶活力

将黄色短杆菌培养在含有延胡索酶的平板培养基上,该菌产生的延胡索酸酶能转化延胡索乱Ｌ－苹果酸。用６Ｎ硫酸处理平板后,其菌落周围便会出现清晰的透明圈。     

HBV─C基因探针的制备及应用

应用ＰＣＲ技术扩增出ＨＢＶＤＮＡＣ基因片段并与ｐＡＴ１５３质粒重组,转化到Ｅ．ｃｏｌｉＲＲＩ中,经体内扩增,提纯,用光生物素标记,制备了Ｃ基因的重组质粒探针。该探针检测灵敏度在Ｓｏｕｔｈｅｒｎ印迹中达１ｐｇ,在点印迹中为５ｐｇ。用此探针以Ｓｏｕｔｈｅｒｎ印迹方式配合ＰＣＲ技术检测乙肝病人血清中的ＨＢＶＤＮＡ,在５３例ＰＣＲ产物电泳检测阴性的样品中,Ｓｏｕｔｈｅｒｎ杂交又检出１８例阳性。