生物转化中新酶应用研究的最新进展

目前,在生物转化中出现了越来越多与传统工艺大不相同的新工艺,特别是生物酶在生物转化中的应用。硫酸酯酶可以立体选择性和对映体选择性地催化水解仲烷基硫酸酯,并通过改变其构象从而产生纯手性的化合物;卤代醇脱卤酶可以催化多种非天然亲核性底物;在水介质中,裂解酶可以催化偶姻或苯偶姻反应从而产生非对称的碳-碳键;最近还出现了用酶催化生产纯手性α-L-氨基酸的新方法。     

Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Stereochemistry-dependent inhibition of RAS farnesylation by farnesyl phosphonic acids

This investigation compares the effects of three farnesyl pyrophosphate analogs on selected aspects of isoprenoid metabolism. E,E-α-Hydroxyfarnesylphosphonate was prepared by an improved variation on a literature synthesis, which also gave access to the new Z,E-α-hydroxyfarnesyl- and α-hydroxygeranylphosphonates. A striking find is that only E,E-α-hydroxyfarnesylphosphonate induces alteration of RAS processing in intact human-derived leukemia cells and inhibits farnesyl protein transferase in enzyme assays, while the Z,E-α-farnesyl- and geranylphosphonates are inactive. The inhibitory activity of E,E-α-hydroxyfarnesylphosphonate is greater in enzyme than intact cell assays. This active compound does not significantly inhibit geranylgeranyl protein transferase I or squalene synthase, nor does it diminish cholesterol synthesis. These results indicate that the length of the terpenoid chain and olefin stereochemistry allow selective inhibition of critical enzymes of terpenoid metabolism. Discrimination was observed between inhibition of farnesyl protein transferase and squalene synthase by E,E-α-hydroxyfarnesylphosphonate, even though both enzymes utilize farnesyl pyrophosphate as their natural substrate.     

Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in α-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 50% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.     

Alicyclobacillus acidocaldarius Squalene‐Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3‐Oxidosqualene

The acyclic molecule squalene ( 1 ) is cyclized into 6,6,6,6,5‐fused pentacyclic hopene ( 2 ) and hopanol ( 3 ; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene‐hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio‐ and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all‐chair conformation. The Ala306 residue in AaSHC is conserved in known squalene‐hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5‐fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair‐chair‐chair‐boat conformation; in contrast, the normal chair‐chair‐chair‐chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)‐2,3‐oxidosqualenes (OXSQs) to generate 3α,β‐hydroxyhopenes and 3α,β‐hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)‐OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.     

Sulfonated poly(ethylene‐ran‐styrene) ionomers

Ionomers were prepared by partially sulfonating the styrene units of a poly(ethylene‐ran‐styrene) (ES) copolymer. The metal salt derivatives of sulfonated ES had higher T_g, melt viscosity and rubbery modulus than did the parent ES because of the formation of labile, physical crosslinks from intermolecular dipole–dipole associations between metal sulfonate groups. Microphase separation of ion‐rich aggregates with a characteristic size of 3–9 nm also occurred as a result of the strong intermolecular associations. Copyright © 2005 Society of Chemical Industry     

Novel zinc complexes with acetyloacetonate, imidazole and thiolate ligands: Crystal structure of a zinc complex of relevance to farnesyl transferase

New zinc(II) complex 1 with three different ligands: acetyloacetonate, tri-tert-butoxysilanethiolate and neutral 2-isopropylimidazole has been obtained and characterized by X-ray diffraction. It is the first example of a mononuclear zinc(II) complex mimicking the active site of farnesyl transferase. (2-Isopropylimidazole)bis(acetylacetonato)zinc(II) 2 is also described as an intermediate product of the reaction leading to the formation of 1.     

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1–geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X‐ray diffraction were carried out by employing an Escherichia coli whole‐cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)‐eremophilene synthase. The very tight binding of (+)‐eremophilene (～0.40 μM ), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1–geoA gene cluster is required for the biosynthesis of (+)‐eremophilene derivatives.     

Chemical Signals of Synthetic Disaccharide Derivatives Dominate Rhamnolipids at Controlling Multiple Bacterial Activities

Microbes secrete molecules that modify their environment. Here, we demonstrate a class of synthetic disaccharide derivatives (DSDs) that mimics and dominates the activity of naturally secreted rhamnolipids by Pseudomonas aeruginosa. The DSDs exhibit the dual function of activating and inhibiting the swarming motility through a concentration‐dependent activity reversal that is characteristic of signaling molecules. Whereas DSDs tethered with a saturated farnesyl group exhibit inhibition of both biofilm formation and swarming motility, with higher activities than rhamnolipids, a saturated farnesyl tethered with a sulfonate group only inhibits swarming motility but promote biofilm formation. These results identified important structural elements for controlling swarming motility, biofilm formation, and bacterial adhesion and suggest an effective chemical approach to control intertwined signaling processes that are important for biofilm formation and motilities.     

Small‐molecule‐induced miscibility of isosorbide‐based polycarbonate with bisphenol A polycarbonate

In this study, we investigated the influence of the small molecule 4,4′‐thiobis(6‐tert‐butyl‐m‐methyl phenol) (AO300) on the miscibility of poly(isosorbide‐co‐1,4‐cyclohexanedimethanol carbonate) (IcC–PC) with bisphenol A polycarbonate (BPA–PC) through the formation of hydrogen‐bonding networks. Differential scanning calorimetry and morphological observation revealed that the initially, immiscible BPA–PC/IcC–PC blends become miscible through the addition of small molecules. Fourier transform infrared spectroscopy confirmed that intermolecular hydrogen bonds formed between the hydroxyl groups of AO300 and the carbonyl groups of the studied polycarbonates. These polycarbonates exhibited different hydrogen‐bonding behaviors and various degrees of glass‐transition temperature composition dependence. Dynamic mechanical analysis demonstrated that AO300 played an antiplasticization role in the BPA–PC/IcC–PC blends with improved storage moduli. To our knowledge, this article is the first to describe the miscibility of isosorbide‐based polycarbonate with BPA–PC. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44537.     

Modification of Chemlok 607 for Bonding Steel to Fluororubber

Chemlok 607 is widely used for bonding metals to fluororubber. However, it is not satisfactory with respect to the bond strength between metals and fluororubber. The aim of this study was to improve adhesion properties of Chemlok 607 for bonding metals to fluororubber. Modifications of Chemlok 607 were carried out with urotropine–resorcinol complexes. The effects of the complexes and vulcanizing pressure on adhesion were studied by tensile lap shear tests and scanning electron microscopy (SEM). Considerably positive adhesion effect was achieved by modification of Chemlok 607 with 1:1 (mole ratio) complex of urotropine and resorcinol when it was used for bonding steel to fluororubber vulcanized by an amine compound; tensile lap shear strength could be achieved to a level of 4.7 to 5.4 MPa. The complex was an ideal bonding accelerant agent for Chemlok 607 because it could promote formation of chemical bonds between fluororubber and Chemlok 607. When incorporated in the vulcanizing process, the complex could also generate many kinds of amino resins with a large number of hydroxide radicals (–OH) which improved adhesion to metal.     

Synthesis of cationic flocculant by radiation-induced copolymerization of methyl chloride salt of N,N-dimethylaminoethyl methacrylate with acrylamide in aqueous solution. II. Copolymerization at higher monomer concentration

The radiation-induced copolymerization of methyl chloride salt of N,N-dimethylaminoethyl methacrylate with acrylamide was studied to prepare a polymer flocculant that can be handled as a solid. The product obtained in the presence of 5–20% water was a solid and could be ground to a powder without drying. In order to obtain a water-soluble polymer at a higher concentration, the effect of various additives on the copolymerization was investigated and found that alcohols bearing a hydrogen atom attached to the tertiary carbon atom effectively inhibit intermolecular crosslinking to give water-soluble polymer. It is suggested that the formation of water-insoluble polymer is predominantly attributable to the crosslinking of polymer chains rather than to the imidation of amide groups. Copolymerization in the presence of isopropyl alcohol as inhibitor of the crosslinking was also studied and compared with that reported previously, which was carried out at a lower monomer concentration without additives.     

Mechanisms of formation of volatile aromatic pyrolyzates from poly(vinyl chloride)

The formation of volatile aromatic pyrolyzates in poly(vinyl chloride) has been studied by pyrolysis–gas chromatography–mass spectroscopy. Results from isotopic labeling experiments with perdeutero-PVC suggest that the aromatic pyrolyzates can be divided into three general classes with respect to their formation mechanisms: (1) intramolecular—benzene and naphthalene are formed almost exclusively via intramolecular cyclization reactions; (2) mixed intra/intermolecular—styrene and indene can be formed either intramolecularly or via transfer of one hydrogen atom between PVC chains; (3) intermolecular—toluene and methylnaphthalene can be formed via transfer of one or two hydrogen atoms between chains. The isotopic distributions observed in the labeling experiments can be reasonably well explained without involving crosslinking (intermolecular C? C bond formation) reactions.     

Mechanism of reaction of cyanogen bromide-activated agarose with amines and the solvolysis of amine ligands

Cyanogen bromide activated agarose is widely employed as the water-insoluble matrix in immunoadsorption and affinity chromatography for the immobilisation of proteins. A reaction mechanism is presented for the cyanogen bromide activation of the galactan, agarose, the coupling of model amines to the activated carbohydrate matrix and the detachment of ligands. Dansyl (Dns) derivatisation of the acid hydrolysed and unhydrolysed amine derivatives resulting from the reaction of cyanogen bromide-activated Sepharose® and N-α-acetyl-L-ornithine reveal the production of arginine, indicating that the amine-Sepharose conjugate is an O-substituted isourea. Thin layer chromatography of the Dns-derivatised unhydrolysed filtrate from the reaction indicated the formation of several derivatives which suggest that the isourea bonds can be cleaved by the solvolytic attack of nucleophiles in the pH range 8–9.5. Nucleophilic attack by amino groups on the amine-Sepharose conjugates releases N-monosubstituted guanidine, N₁, N₂ disubstituted ornithine and substituted ureas.     

共结晶分离技术研究进展

使用共结晶技术分离有机小分子,特别是一些不能成盐的、高纯度要求的原料药(API)的分离是晶体工程应用的前沿。其原理是通过分子间的识别作用形成共晶,改变目标分子的晶格能或溶解特性,从而实现分离。针对共结晶在分离中的应用,本文从共结晶分离的热力学原理出发,系统综述了共结晶在分离纯化非手性分子以及手性API及中间体中的实例。从分子结构、分子间的相互作用力、溶度积常数、溶剂体系等不同角度对分离实例进行分析。针对该技术现存的问题如共晶形成物的选择缺乏规律性,实际API纯化体系的复杂性,共晶形成物回收利用的可行性,指出建立系统的共晶形成物选择方法、深入地研究热力学行为是未来的主要研究方向。     

Photometric Characterization of the Reductive Amination Scope of the Imine Reductases from Streptomyces tsukubaensis and Streptomyces ipomoeae

Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C₄–C₈) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94–96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source.     

A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto

Bacillus subtilis subsp. natto secretes the ComX_natto pheromone as a quorum‐sensing pheromone to produce poly‐γ‐glutamate for biofilm formation. The amino‐acid sequence of the pheromone is Lys‐Trp‐Pro‐Pro‐Ile‐Glu, and the tryptophan residue is post‐translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C‐terminal end of the precursor peptide ComX_natto. Here, we report the functional analysis of ComQ_natto, which catalyzes a unique farnesyl‐transfer reaction. ComQ_natto recognizes not only full‐length ComX_natto but also N‐ and/or C‐terminal truncated ComX_natto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQ_natto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.     

Glass‐transition behavior of poly(methacrylonitrile)

The glass‐transition temperatures (T_g) of some polymers reported in the literature have always been a source of great uncertainty. The values reported for poly(methacrylonitrile) (PMAN, 100 and 120°C) are well above the value determined in this study (67°C). It is clearly shown by FTIR and DSC work that formation of cyclic structures during the drying of PMAN, even at low temperatures, is the main reason for the high T_g values observed. The contributions of naphthydrine type cyclic structures and intermolecular crosslinks in the increase of the T_g are determined over an aging temperature interval of 90–300°C. The combined effects of intra‐ and intermolecular linking cause an increase in the T_g from 67 to 116°C. The hardness measurements also confirm the value determined by DSC. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 1936–1943, 2001     

Untersuchung und lebensmittelrechtliche Beurteilung von raffinierten und nicht raffinierten pflanzlichen Speiseölen und Fetten

Investigation and Legal Food Estimation of Refined and Non-refined Vegetable Oils and Fats The rules of the German foodbook defines a native and coldpressed oil as a product which is not treated by alkali, bleaching earth or water vapor. A study was carried out to find an analytical criterion to differ vapor-treated from non-refined oils. We investigated the formation of trans-isomers of linoleic acid as well as the alteration of other constituents (sterols, tocopherols) by means of capillary GLC and HPLC.