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The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus‐guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3. Upon phylogenetic analysis of a set of distantly related P450s (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature (Topt) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.  相似文献   
3.
In this study, we report that Streptomyces asterosporus DSM 41452 is a producer of new molecules related to the nonribosomal cyclodepsipeptide WS9326A and the polyketide annimycin. S. asterosporus DSM 41452 is shown to produce six cyclodepsipeptides and peptides, WS9326A to G. Notably, the compounds WS9326F and WS9326G have not been described before. The genome of S. asterosporus DSM 41452 was sequenced, and a putative WS9326A gene cluster was identified. Gene‐deletion experiments confirmed that this cluster was responsible for the biosynthesis of WS9326A to G. Additionally, a gene‐deletion experiment demonstrated that sas16 encoding a cytochrome P450 monooxygenase was involved in the synthesis of the novel (E)‐2,3‐dehydrotyrosine residue found in WS9326A and its derivatives. An insertion mutation within the putative annimycin gene cluster led to the production of a new annimycin derivative, annimycin B, which exhibited modest inhibitory activity against Plasmodium falciparum.  相似文献   
4.
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p H值降低的速率依次为:L4B5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(P0.05)。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。  相似文献   
5.
The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.  相似文献   
6.
The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.  相似文献   
7.
Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.  相似文献   
8.
Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted ‘energetic coupling’ describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein–ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.  相似文献   
9.
脉冲强光对啤酒酵母的诱变效应   总被引:1,自引:0,他引:1  
张佰清  孙栏梦 《食品科学》2015,36(7):153-157
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。  相似文献   
10.
泰乐菌素高产菌株的筛选   总被引:1,自引:0,他引:1  
以弗氏链霉菌(Streptomyces fradiae)TP-116菌株作为出发菌株,采用紫外线、氯化锂和微波三重复合诱变,通过泰乐菌素抗性定向筛选,获得一株高产菌株YBT-06,并对其进行了遗传稳定性试验,结果表明突变株YBT-06生产代谢旺盛,遗传稳定性好,发酵效价比出发菌株提高了25%,达到11066μg/m L。  相似文献   
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