耐高浓度甘油的1,3-丙二醇生产菌的诱变选育

利用紫外线对克雷伯杆菌进行诱变,筛选出耐高浓度甘油且1,3-丙二醇(1,3-PD)产量较高的突变株KpⅥ.实验结果表明,在距离34 cm,功率30 W的紫外灯垂直照射下,最佳紫外诱变时间为6 min,此时,致死率为90.9%.克雷伯杆菌经过紫外诱变后,菌落明显增大,是原始菌株的2~4倍;变异菌株KpⅥ经过6代遗传稳定性考察,甘油消耗率和1,3-PD产量稳定,且保持了较高水平.在甘油质量浓度为90 g.L-1的条件下,变异菌株KpⅥ的甘油消耗率为98.3%,甘油转化率为50.4%,1,3-PD产量为44.81 g.L-1,生产能力达到0.75 g.(L.h)-1.     

Importance of NPA motifs in the expression and function of water channel aquaporin-1

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and effi-cient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 de-letion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.     

Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase

The CphA metallo--lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.Received 3 March 2003; received after revision 4 August 2003; accepted 25 August 2003     

紫外线诱变康宁木霉提高酶活力的研究

通过对康宁木霉菌种的诱变、筛选和固体发酵培养,研究了培养基、培养时间、诱变时间及pH值对诱变菌所产酶的活力的影响．结果表明,康宁木霉经诱变5min,在pH为6．0的玉米秸PDA培养基上培养72h,产生的C1酶和Cx酶的酶活力较高,C1酶的酶活力由73u／g提高到176u／g,增加了2．41倍,Cx酶的酶活力由798u／g提高到2069u／g,增加了2．59倍．     

不对称还原苯乙酮酸甲酯的生物转化菌种选育及条件优化

选用酵母菌、乳酸菌、粪链球菌、假丝酵母等4个种属共15株菌株,研究其对苯乙酮酸甲酯的不对称还原催化特性.以具有R(-)-扁桃酸甲酯较高转化活力的菌株S.cNo.1、S.cNo.3、S.cNo.9作为出发菌株,采用紫外与微波复合诱变的方法,筛选获得S.c3.5.16突变株.考察培养温度、pH值对该突变株转化活力的影响,结果表明,S.c3.5.16菌株的R(-)-扁桃酸甲酯生物转化最适反应条件为培养基pH6.5、温度38℃,苯乙酮酸甲酯转化为扁桃酸甲酯的得率达到99.3%,R(-)-扁桃酸的光学纯度达到95.5%e.e..     

豆凝乳酶产生菌的筛选及诱变育种

从１０４份土样中分离到一株产豆凝乳酶的芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）,酶活力０．３ｕ／ｍｌ。经ＵＶ－ＤＥＳ诱变及培养基调整后,酶活可达１．６ｕ／ｍｌ以上,其最适产酶培养基组成：麦麸２．５％,（ＮＨ４）２ＳＯ４ ０．５％（ＮＨ４）２ＨＰＯ４ ０．５％,ＣａＣｌ２ ０．０５％。     

Mutational effect of the “- 35” element of sorghumpsbA gene promoter

Mutations of the first position T and the third position G in TTGACA, the " - 35" element of sorghum psbA gene promoter, were induced using chemically synthesized 20 nt oligonucleotide primer. Three mutants were produced: ATTACA, GTGACA, and ATGACA. Then the protein binding affinity of the mutants and the wild type sorghum psbA gene promoter was tested in a spinach chloroplast protein extract system. Gel retardation assay of the wild type showed a strong protein-binding band. On the other hand, the protein-binding band of the mutant resulting from single base mutation, ATGACA or GTGACA, showed reduced intensity, while that of the mutant resulting from double base mutation, ATTACA, showed increased intensity. It is thus shown that the " - 35" element plays an important role in controlling the binding between psbA gene promoter and the specific chloroplast proteins; mutation of a single base may exert a substantial influence on the binding affinity.     

mRNA二级结构对重组人白血病抑制因子(rhLIF)在大肠杆菌中表达的影响

真细菌中翻译起始效率通常是由翻译起始位点两侧的ｍ ＲＮＡ 区域二级结构的稳定性决定的．这种稳定性与该区域ＲＮＡ 二级结构的发生自由能（ΔＧ０ ）相对应．重组人白血病抑制因子（ｒｈＬＩＦ）为具有诱导白血病细胞分化、调节骨组织的生长代谢、维持胚胎干细胞的基本特征、促进神经元的分化等广泛生物学活性的细胞因子．为了获得高表达的ＬＩＦ蛋白,通过翻译起始区的位点专一突变,把具有优选密码子和能量优势的ｌｉｆ 片段克隆入ｐＪＬＡ５０３ 载体,然后转化至大肠杆菌中表达,得到表达量提高１０ 倍的重组ＬＩＦ蛋白     

NTG对侧耳的诱变效应

采用正交试验设计以菌落直径为指标研究了NTG对侧耳8405的诱变效应.实验结果表明:当NTG的最终浓度为300μg/mL,处理前孢子先培养14h,于24℃黑暗条件下处理40min时,突变率和正变率高,变异幅度大.