CD59活性位点相关性基因突变的构建与表达

构建了野生型和突变型CD59重组质粒,建立了高效真核表达系统,探讨了W40位点的生物学活性。采用基因点突变技术使CD59W40基因位置缺失（突变1,M1）及C39W40K41→W39W40W41（突变2,M2）,重叠延伸PCR（overlap extension PCR）定点诱变扩增突变基因,重组入真核表达质粒pIRES,利用阳离子脂质体（Lipfectamine2000）将重组质粒转染中国仓鼠卵巢细胞（CHO）进行表达。酶切鉴定及序列测定证实成功构建了pIRES-MICD59、pIRES—M2CD59和pIRES-WTCD59,突变基因约500bp。G418筛选出了CHO转染细胞的稳定细胞克隆,免疫荧光、ELISA检测筛选MICD59、M2CD59和WTCD59蛋白高表达株,连续传代30代有高表达;补体溶细胞反应显示与野生型CD59相比,突变型M1CD59失去对补体的抑制功能,而M2CD59抗补体活性略增高。证实CD59的W40位点对其功能具有重要作用,封闭此位点可提高补体活性,有望用于肿瘤治疗。     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

定点突变提高食品新型L-天冬酰胺酶的活力及热稳定性

L-天冬酰胺酶（L-asparaginase II,EC 3.5.1.1）可将L-天冬酰胺转化为L-天冬氨酸,减少高温加工食品中丙烯酰胺的形成,因而受到人们的广泛关注。该酶在食品加工及预处理阶段的使用已受到人们广泛的关注,但是由于食品加工及预处理过程中环境的复杂性,对L-天冬酰胺酶的性质、热稳定性和酶活等方面有较高的要求。通过序列对比和同源模拟对嗜热菌Pyrococcus yayanosii CH1来源的编码L-天冬酰胺酶的基因PyAsnase进行了3个位点的突变,并在Bacilus subtilis 168 中进行表达,提高了该酶的热稳定性及比酶活。其中突变株E22K较原始菌株相比所得突变体比酶活提高了约37.3%,突变株R111L较原始菌株相比所得突变体的比酶活提高了约31.1%,突变株M92A较原始突变菌株相比所得突变体在85 ℃时的半衰期延长了约30 min。本研究结果为探索L-天冬酰胺酶结构和功能的相互关系提供了借鉴,提高了其在食品工业中的应用前景。     

激光诱变滇"三角大香糯"的遗传效应研究

2 0 0 1年我们用He -Ne激光辐照加电场、磁场激发对滇“三角大香糯”进行了育种研究〔1〕,并得到了五株有较优变异的稻种 (第 1代M1 )。 2 0 0 2年我们种植了这五株稻种 ,并进行了各生育期的田间试验观测。分析了诱变滇三角香糯育出稻种的遗传性质     

植物乳杆菌发酵萝卜干品质变化分析

为加快发酵速率,降低产品亚硝酸盐含量,并提高产品品质,本研究以萝卜干为原料,分别接种植物 乳杆菌L4（Lactobacillus plantarum L4）和植物乳杆菌B5（L. plantarum B5）,并以自然发酵为对照,萝卜干 发酵时间为56 d,研究L4和B5对萝卜干品质的影响。结果表明：L4、B5和自然发酵pH值降低的速率依次为： L4＞B5＞自然发酵。亚硝酸盐含量随着发酵时间的延长先增加后减小,其中L4发酵在22 d左右出现亚硝酸盐 峰,峰值为（3.23±0.17）mg/kg,B5和自然发酵在33 d左右出现亚硝酸盐峰,峰值分别为（2.04±0.12）mg/kg和 （3.79±0.25）mg/kg（P＜0.05）。挥发酯含量、游离氨基酸含量都随着发酵时间的延长呈上升趋势。L*、b*随着 发酵时间的延长呈下降趋势,而a*随着发酵时间的延长呈上升趋势。发酵结束时,L4、B5和自然发酵感官评分别 为88.7±2.56、81.8±1.49和74.1±3.88。由此表明,植物乳杆菌L4和B5可以缩短萝卜干的发酵周期,提高萝卜干安 全性和品质,其中L4表现比B5好。     

脉冲强光对啤酒酵母的诱变效应

采用脉冲强光对啤酒酵母菌种进行诱变处理,脉冲处理电压分别为1 000、1 500、2 000、2 500、3 000 V,闪照次数分别为4、8、16、32、48。测定出发菌株和筛选出的变异菌株的凝聚性、双乙酰产量、发酵速率、发酵结束理化性质等指标,比较变异菌株与出发菌株综合指标的差异。结果表明：经过初筛和复筛,筛选出10#和12#两株发酵性能较好的菌株。脉冲强光诱变处理并未对啤酒酵母的发酵度产生负面效应,而是有所提高或保持原酵母菌株的优良发酵性能。     

常压室温等离子体生物诱变育种及其应用研究进展

大气压射频辉光放电（RF APGD）等离子体具有大气压下操作不需要真空系统、气体温度低、活性粒子浓度高、放电均匀性好、可控性强等特点,能够与各类生物分子发生作用,在生物技术中的应用受到广泛的关注。本研究团队将RF APGD等离子体射流引入生物诱变育种领域,对其物理特性及其与生物大分子和整细胞的作用机制进行了系统研究,并将其开发成新一代高效诱变育种仪,命名为常压室温等离子体（ARTP）诱变育种仪。实践证明,ARTP诱变育种仪具有对操作者安全、环境友好、操作简便、突变快速、突变率高、获得的突变体性状稳定等特点,目前已成功应用于包括细菌、放线菌、真菌、酵母、微藻等在内的四十余种微生物的诱变育种。本文将对ARTP生物育种技术的最新研究进展进行综述,以期ARTP快速生物突变技术在生物进化研究及工业生物菌种改造上发挥重要作用。     

Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-NADH

Well-conserved three consecutive Pro residues (Pro247–249) in the NADH-binding subdomain of NADH-cytochrome b₅ reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coli. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6×His-tag sequence at the NH₂-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH:ferricyanide reductase and NADH:cytochrome b₅ reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease k_cat with slight increase in K_m for the physiological electron donor NADH. However, K_m values for the electron acceptors (both cytochrome b₅ and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b₅.     

In vitro evolution of proteins

Consecutive rounds of diversification and selection of the fittest is believed to be the main driving force for the evolution of life. For the evolution of life to proceed, all living cells are surrounded by a lipid bilayer that separates their own genes from the external environment and from those of other organisms. In this way, the genetic information of an individual is replicated on the basis of their phenotype; thus the enrichment of the fittest will occur. Hence, evolution is based on linkage between genotype and phenotype owing to the surrounding of the genetic material with a barrier. The linkage between genotype and phenotype is also known to be essential for the directed evolution of proteins. Indeed, systems for molecular evolution, including phage display, ribosome display, and in vitro compartmentalization, all satisfy this requirement in different ways. These systems have been shown to be powerful tools for high-throughput screening for the functions of proteins, screening as many as <10(12) molecules in 1 d. These selection systems in combination with various gene libraries yield proteins with improved or altered biophysical properties, and may even allow the generation of proteins with novel functions.     

Enzymatic activity of Campylobacter jejuni hippurate hydrolase

The hippurate hydrolase enzyme of Campylobacter jejuni was expressed in Escherichia coli as a six-histidine-tagged fusion protein. The purified recombinant enzyme was characterized to gain an understanding of the structure and activity of the hippurate hydrolase. The recombinant enzyme had a native molecular mass of 193+/- 11 kDa a reduced molecular mass of 42.4+/- 0.8 kDa, and possessed 1.98+/- 0.68 molecules of zinc per enzyme subunit molecule, suggesting that it was a homotetramer with two associated zinc ions. The enzyme was a metallocarboxypeptidase that was sensitive to silver, copper and ferrous ions, and displayed optimal activity at pH 7.5 and 50 degrees C. It hydrolyzed carboxypeptidase substrates in vitro, displaying its highest activity against N-benzoyl-linked small aliphatic amino acids. A high proportion of the enzyme structure consisted of highly ordered alpha-helix and beta-sheet sequences. An alignment of the amino acid sequence of the hippurate hydrolase enzyme with those of related enzymes with similar activities revealed several conserved amino acids, which might be involved in enzyme catalysis or metal ion binding for the enzyme. Site-directed mutagenesis of the recombinant enzyme demonstrated that the Asp(76), Aps(104), Glu(134), Glu(135), His(161) and His(356) positions were important for the catalytic activity of the enzyme.