酸催化蔗糖水解的动力学研究

利用旋光度与蔗糖反应体系浓度的关联,研究了盐酸、硫酸、磷酸、醋酸等不同酸催化剂以及不同浓度的盐酸在不同温度下对蔗糖水解反应速率的影响。结果表明,不同酸催化剂对蔗糖水解起催化作用的是氢离子;[H]+从3 mol/L变化到4 mol/L时,蔗糖水解反应从一级反应近似转变为二级反应;催化剂降低了反应的活化能,加快了反应速率;酸催化蔗糖水解最佳条件是盐酸,浓度为3 mol/L,反应温度为45℃。     

无溶剂法合成蔗糖硬脂酸酯的工艺研究

以乙醇代替有毒的甲醇与硬脂酸在酸催化下进行反应制出了硬脂酸乙酯，以硬脂酸乙酯与蔗糖为原料在催化剂和乳化剂的作用下，进行酯交换反应合成出蔗糖硬脂酸酯，本文详细考察了该合成反就过程中乳化剂，催化剂用量，反应温度，原料配比，反应时间等因素对蔗糖硬脂酸酯产率的影响，得出了较佳合成了工艺条件，即硬脂酸乙酯：蔗糖摩尔比为2．5：1，乳化剂与蔗糖质量比为0．1：1，催化剂与反应物料质量经为0．14：1，反应温度120℃，压力4.35kPa，时间3.5h，蔗糖酯产率约75．2％，此外，还把蔗糖硬脂酸酯制成易挥发衍生物，建立了分析其组成的气相色谱方法。     

酸催化水解蔗糖制取乙酰丙酸

以蔗糖为原料,在H2SO4催化下制备乙酰丙酸,主要考察了反应时间、反应温度、蔗糖浓度、H2SO4浓度对乙酰丙酸产率的影响。实验结果表明,反应时间60min,反应温度110℃,蔗糖浓度0.4mol/L,H2SO4浓度3.5mol/L条件下乙酰丙酸的产率最高。     

Noncanonical Reactions of Flavoenzymes

Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.     

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid- and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta.     

Self-Assembly of Proteinaceous Shells around Positively Charged Gold Nanomaterials Enhances Colloidal Stability in High-Ionic-Strength Buffers

The enzyme lumazine synthase (LS) has been engineered to self-assemble into hollow-shell structures that encapsulate unnatural cargo proteins through complementary electrostatic interactions. Herein, we show that a negatively supercharged LS variant can also form organic–inorganic hybrids with gold nanomaterials. Simple mixing of LS pentamers with positively charged gold nanocrystals in aqueous buffer spontaneously affords protein-shelled gold cores. The procedure works well with differently sized and shaped gold nanocrystals, and the resulting shelled complexes exhibit dramatically enhanced colloidal stability over a wide range of pH (4.0–10.0) and at high ionic strength (up to 1 m NaCl). They are even stable over days upon dilution in buffer. Self-assembly of engineered LS shells in this way offers an easy and attractive alternative to commonly used ligand-exchange methods for stabilizing inorganic nanomaterials.     

Solid-Phase Synthesis of Substrate-Based Dipeptides and Heterocyclic Pseudo-dipeptides as Potential NO Synthase Inhibitors

More than 160 arginine analogues modified on the C-terminus via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side-chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side-chain thiourea group was either let unchanged, S-alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S-Et- or a S-Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-oxadiazole series were shown to inhibit nNOS and iNOS with IC₅₀ in the 1–50 μM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with similar efficiency than the reference compounds L-NIL and SEIT.     

细菌纤维素合成与鉴定研究综述

细菌纤维素（BC）因其独特性能被广泛应用于医药、食品等领域,目前其高产量菌株筛选、合成成本降低及合成途径改良等成为研究热点。本文依据国内外文献并结合团队研究成果对BC合成与鉴定的相关研究进行综述。首先对BC合成菌筛选及碳源利用的研究进行了分析,总结了降低BC合成成本的研究思路。其次对鉴定菌株合成产物的方法进行了归纳,总结了不同方法的特点。然后结合本团队筛选出的BC生产菌XJL-06-4 BC合成酶基因分析结果,综述了BC合成途径、合成酶存在形式以及基因水平调控作用,为BC在分子水平上通过改变合成途径提高产量提供新思路。最后,总结BC微生物发酵生产存在的问题,多角度提出解决方案。     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.