Solid‐Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

Directed evolution of stereo‐ and regioselective enzymes as catalysts in organic chemistry and biotechnology constitutes a complementary alternative to selective transition‐metal catalysts and organocatalysts. Saturation mutagenesis at sites lining the binding pocket has emerged as a key method in this endeavor, but it suffers from amino acid bias, which reduces the quality of the library at the DNA level and, thus, at the protein level. Chemical solid‐phase gene synthesis for library construction offers a solution to this fundamental problem, and the Sloning and Twist platforms are two possible options. This concept article analyzes these approaches and compares them to traditional PCR‐based saturation mutagenesis; the superior commercial Twist technique shows almost no bias.     

Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity

Substrate‐promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio‐ and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)‐lisofylline, a compound under investigation for its anti‐inflammatory properties, was chosen as a first proof‐of‐principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.     

Mutation of Conserved Residues Increases in Vitro Activity of the Formylglycine‐Generating Enzyme

The formylglycine‐generating enzyme (FGE) recognizes proteins with a specific cysteine‐containing six‐amino‐acid motif and converts this cysteine residue into formylglycine. The resulting aldehyde function provides a unique handle for selective protein labeling. We have identified two mutations in FGE from Thermomonospora curvata that increase this catalytic efficiency more than 40‐fold. The resulting activity and stability, as well as its ease of recombinant production, make this FGE variant a practical reagent for in vitro protein engineering.     

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。     

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.