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51.
Xiaoyan Guo Yunfang Gao Fangzheng Liu Yong Tao Haibo Jin Jianjun Wang Sheng Wu 《Microbial biotechnology》2023,16(6):1333-1343
R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales. 相似文献
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Alterations in the amount of fibronectin and in the number of its receptors during myoblast differentiation of chicken embryo were investigated. The amount of fibronectin in the cell surface pool as measured by immunoblotting decreased during myogenesis To identify and characterize the fibronectin receptors on the myoblasts, the interactions of the 28,000 dalton (28 kDa) amino terminal fragment and 85,000 dalton (85 kDa) cell-binding fragment of fibronectin with my-oblasts were examined. The binding of the 28 kDa fragment was found to be time-dependent and reached a maximum level within 60 min. The unlabeled 28 kDa fragment inhibited the binding of the radioiodinated 28 kDa fragment, whereas the unlabeled 85 kDa fragment and antibody to integrin did not inhibit it, suggesting that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. There was a single class of 3.4 × 105 binding sites per cell with an apparent dissociation constant of 1.4 × 10?7 M on 30 hr old myoblasts. The specific binding of the radioiodinated 28 kDa fragment to myoblasts decreased as the fusion proceeded. This decrease of binding was consistent with the decrease in the amount of fibronectin. Furthermore, the levels of fibronectin and binding of the radioiodinated 28 kDa fragment in the fusion-blocked myoblasts by EGTA treatment appeared to remain constant. These results suggest that the decrease and/or loss of fibronectin during myoblast fusion is closely correlated with the alteration of fibronectin receptors and with the fusion of myoblasts. 相似文献
54.
利用改进的kar交配法,将一个含有340kb人基因组DNA的YAC片段的供体酵母菌株YAC23与受体菌株YLB504进行交配,以选择平板对所形成的候选YAC导入菌进行筛选。经PCR分析,候选YAC导入菌在404bp处有一个扩增带,即具有受体菌株的交配型(MAT α)。进一步用脉冲电泳进行核型鉴定,证实YACs己成功地进入受体,实现了YACs从一个宿主到另一个宿主之间的转移。 相似文献
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植酸对稻米品质影响的研究(Ⅰ) 总被引:2,自引:0,他引:2
本文报道在水稻生长的抽穗期、齐穗期、灌浆期和蜡熟期喷施不同浓度植酸改良稻米品质的研究。结果表明,在水稻生长的不同阶段,控制喷施植酸浓度可提高糙米率,降低稻米垩白。 相似文献
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应用离体叶片法,对9个棉花种质进行了鉴定,试验结果表明;种质间抗生性和忌避性差异显著;同株棉花不同部位的叶片对朱砂叶螨的抗生性无显著性差异。通过对叶螨在不同棉花种质上种群增长动态进行系统聚类,可将9个棉花种质划分为3类:斯字棉825-91、杞县86789、鄂棉314、苏联8911为1类,中棉164、潼南接龙棉、新库861517-2、南农NAC90-2为1类,美棉7-15独立为1类。依据朱砂叶螨在不同种质上的种群增长曲线和高峰期螨量增长倍数,可将9个种质划分为3个类型;斯字棉825-91、新库861517-2为抗性类型,潼南接龙棉、美棉7-15、南农NAC90-2为感性类型,其余为中抗类型。从忌避性看:斯字棉825-91、美棉7-15表现出较高的忌避性。 相似文献
60.
Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn2+, methione, and other sulfur-containing amino acids and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5–20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 ± 2 kDa subunits. CSAD appears to require Mn2+ for its maximum activity. Other divalent cations fail to replace Mn2+ in activation of CSAD activity. However, the precise role of Mn2+ in the action of CSAD remains to be determined. 相似文献