一种新型微生物卤醇脱卤酶的研究进展

卤醇脱卤酶是细菌降解环境中重要污染物有机卤化物的关键酶之一,具有与其他已知脱卤酶不同的脱卤机制。它是一类通过分子内亲核取代机制催化邻卤醇转化为环氧化物的脱卤酶,可以高效催化有机邻卤醇进行脱卤反应,在治理环境污染方面具有十分重要作用。此外,在催化环氧化物和邻卤醇之间的转化反应中,卤醇脱卤酶具有很高的立体选择性,因而在手性药物合成方面也有广阔的应用前景。我们着重从卤化物生物降解途径、脱卤机制及应用等方面介绍了卤醇脱卤酶的最新研究进展,同时对卤醇脱卤酶改造的新方法进行了阐述与展望。     

A new catalytic function of halohydrin hydrogen-halide-lyase, synthesis of beta-hydroxynitriles from epoxides and cyanide.

Halohydrin hydrogen-halide-lyase, which catalyzes the interconversion of halohydrins to epoxides, purified from a recombinant E. coli was found to catalyze the transformation of 1,2-epoxybutane into beta-hydroxyvaleronitrile in the presence of cyanide. Chloride inhibited competitively the formation of beta-hydroxyvaleronitrile. The enzyme also catalyzed the transformation of some other epoxides into the corresponding beta-hydroxynitriles in the presence of cyanide.     

Identification and characterization of a highly S-enantioselective halohydrin dehalogenase from Tsukamurella sp. 1534 for kinetic resolution of halohydrins

Halohydrin dehalogenases are remarkable enzymes which possess promiscuous catalytic activity and serve as potential biocatalysts for the synthesis of chiral halohydrins, epoxides and β-substituted alcohols. The enzyme HheC exhibits a highly R enantioselectivity in the processes of dehalogenation of vicinal halohydrins and ring-opening of epoxides, which attracts more attentions in organic synthesis. Recently dozens of novel potential halohydrin dehalogenases have been identified by gene mining, however, most of the characterized enzymes showed low stereoselectivity. In this study, a novel halohydrin dehalogenase of HheA10 from Tsukamurella sp. 1534 has been heterologously expressed, purified and characterized. Substrate spectrum and kinetic resolution studies indicated the HheA10 was a highly S enantioselective enzyme toward several halohydrins, which produced the corresponding epoxides with the ee (enantiomeric excess) and E values up to >99% and >200 respectively. Our results revealed the HheA10 was a promising biocatalyst for the synthesis of enantiopure aromatic halohydrins and epoxides via enzymatic kinetic resolution of racemic halohydrins. What’s more important, the HheA10 as the first individual halohydrin dehalogenase with the highly S enantioselectivity provides a complementary enantioselectivity to the HheC.     

Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.     

Chemistry of glucal halohydrins(II): An unusual protecting group effect in the competitive formation of formyl furanosides and methyl glycosides

A remarkable protecting group influence was observed in the base-induced reaction of protected halohydrins derived from -glycals. Tri-O-methyl and tri-O-benzyl halohydrins react with cesium carbonate in methanol at room temperature to give methyl glycosides as the major product and unsaturated formyl furanosides as the minor product. Whereas, the tri-O-tert-butyldimethylsilyl (t-BuMe₂Si)-protected halohydrins reacted with cesium carbonate in methanol at room temperature to give a mixture of epimeric formyl furanosides, and at reflux to give an unsaturated formyl furanoside, as the only products. The tri-O-methyl and tri-O-benzyl halohydrins react slowly at elevated temperature to give predominantly furans. In comparison, the tri-O-t-BuMe₂Si halohydrins reacted completely after five minutes to give a mixture of epimeric formyl furanosides. The tri-O-t-BuMe₂Si iodohydrins were oxidized to the corresponding iodolactones, which also underwent a based-induced ring contraction in methanol to give the furanose 1-methylcarboxylate esters.     

Enantioselectivity of Epoxide Formation from Halohydrins by Means of Flavobacterium Rigense

The formation of epoxides from several halohydrins was achieved using resting cells from Flavobacterium rigense. The reaction showed a high substrate specificity for halohydrins with a terminal halogen atom but only low enantioselectivity (12-58% e.e.). The epoxides always had the (S)-configuration. Substrates which in the halogen atom was replaced by another leaving group (-O-SO₂CH₃, -O-Tos, -N₃) were not accepted. An attempt to improve the enantioselectivity by using a two phase system consisting of an aqueous and an organic solvent phase was not successful.     

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.     

Enantioselectivity of Epoxide Formation from Halohydrins by Means of Flavobacterium Rigense

The formation of epoxides from several halohydrins was achieved using resting cells from Flavobacterium rigense. The reaction showed a high substrate specificity for halohydrins with a terminal halogen atom but only low enantioselectivity (12–58% e.e.). The epoxides always had the (S)-configuration. Substrates which in the halogen atom was replaced by another leaving group (-O-SO₂CH₃, -O-Tos, -N₃) were not accepted. An attempt to improve the enantioselectivity by using a two phase system consisting of an aqueous and an organic solvent phase was not successful.     

唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

A novel and efficient enzymatic method for the production of peptides from unprotected starting materials

We report herein the development of a novel and efficient enzymatic method for the production of oligopeptides. This newly discovered method is a simple, cost-effective process, using unprotected amino acids as substrates in an aqueous solution and producing peptides in high yield. The target of our initial screen was l-alanyl-L-glutamine, a dipeptide of significant industrial interest by virtue of its widespread use in infusion therapy. By means of the screening of microorganisms that can catalyze the peptide-forming reaction producing l-alanyl-L-glutamine from L-alanine methylester (acyl donor) and L-glutamine (nucleophile), we discovered that Empedobacter brevis ATCC 14234 produced l-alanyl-L-glutamine most efficiently. The newly found enzyme purified from E. brevis ATCC 14234 facilitates significantly high production yields of l-alanyl-L-glutamine from L-alanine methylester and L-glutamine in an aqueous solution--more than 80% yield based on L-alanine methylester. In addition, this enzyme has wide substrate specificity--both for acyl donors and nucleophiles--and can catalyze peptide-forming reactions not only to produce various dipeptides from the corresponding amino acid esters and amino acids but also to produce various oligopeptides from the corresponding amino acid esters and peptides.     

Isolation of a bacterium assimilating (R)-3-chloro-1,2-propanediol and production of (S)-3-chloro-1,2-propanediol using microbial resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.     

Opioid Peptides from Human β-Casein

A bacterium that assimilates 2,3-dichloro-1-propanol was isolated from soil by enrichment culture. The strain was identified as Pseudomonas sp. by the taxonomic studies. The strain converted 2,3-dichloro-1-propanol to 3-chloro-1,2-propanediol, releasing chloride ion. The conversion was stereospecific because the residual 2,3-dichloro-1-propanol and formed 3-chloro-1,2-propanediol gave optical rotation. The resting cells converted various halohydrins to the dehalogenated alcohols, and cell-free extracts had strong epoxyhydrolase activity. These results indicated that the strain assimilated 2,3-dichloro-1-propanol via 3-chloro-1,2-propanediol, glycidol, and glycerol. The possibility to manufacture optically active 2,3-dichloro-1-propanol is discussed.     

Chiral C3 epoxides and halohydrins: Their preparation and synthetic application

Epichlorohydrin, 3-chloro-1,2-propanediol and glycidol are versatile chiral C3 syntons in organic synthesis. For the production of these, more efficient and more practical methods are required and many studies have been reported. In this review, biological and synthetic preparations of their synthons and microbial dehalogenation of halohydrins connected with the biological methods are described. Several of their synthetic applications are also introduced.     

Features and applications of bacterial sialidases

Sialidases, or neuraminidases (EC 3.2.1.18), belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate residues from the glycans of glycoproteins, glycolipids, and polysaccharides. In bacteria, sialidases can be used to scavenge sialic acids as a nutrient from various sialylated substrates or to recognize sialic acids exposed on the surface of the host cell. Despite the fact that bacterial sialidases share many structural features, their biochemical properties, especially their linkage and substrate specificities, vary widely. Bacterial sialidases can catalyze the hydrolysis of terminal sialic acids linked by the α(2,3)-, α(2,6)-, or α(2,8)-linkage to a diverse range of substrates. In addition, some of these enzymes can catalyze the transfer of sialic acids from sialoglycans to asialoglycoconjugates via a transglycosylation reaction mechanism. Thus, some bacterial sialidases have been applied to synthesize complex sialyloligosaccharides through chemoenzymatic approaches and to analyze the glycan structure. In this review article, the biochemical features of bacterial sialidases and their potential applications in regioselective hydrolysis reactions as well as sialylation by transglycosylation for the synthesis of sialylated complex glycans are discussed.     

Switch between tyrosinase and catecholoxidase activity of scorpion hemocyanin by allosteric effectors

Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

In vitro selection of kinase and ligase deoxyribozymes

Exploration of the limits of biocatalysis has led to the discovery that DNA has significant potential for enzymatic function. This makes possible the construction of DNA enzymes or "deoxyribozymes" for catalyzing various chemical reactions that could be used to address fundamental questions in biocatalysis or that could find unique applications in biotechnology. Of significant interest are self-modification reactions, given the fundamental role that DNA serves in modern living systems. Recently, in vitro selection strategies have been used to isolate prototypical ATP-dependent deoxyribozymes from random-sequence populations of DNA that catalyze DNA phosphorylation and others that catalyze DNA adenylation. In nature, protein enzymes such as T4 DNA kinase and T4 DNA ligase catalyze identical chemical reactions. These findings suggest that DNA constructs could be engineered to efficiently catalyze other self-modifying reactions, including ATP-dependent DNA ligation. This article provides a detailed overview of the methods used to isolate deoxyribozymes that promote ATP-dependent DNA ligation.     

脂肪酶研发态势的全球专利分析

脂肪酶作为一种能够催化各种酯键断裂和形成的绿色环保生物催化剂,已被广泛应用于食品、饲料、洗涤剂、制药、精细化学以及生物修复等领域。基于智慧芽数据库（Pat Snap）对2020年10月前全球范围内各个国家及地区的脂肪酶相关专利进行统计,分析脂肪酶的技术分布、发展状况和未来趋势,并对比分析我国的脂肪酶研发现状,以期为脂肪酶技术及产业发展方向提供参考依据。     

Mechanistic studies on acyl-transferase ribozymes and beyond

In vitro selection has allowed the isolation of many new ribozymes that are able to catalyze an ever-widening array of chemical transformations. Mechanistic studies on these selected ribozymes have provided valuable insight into the methods that RNA can invoke to overcome different catalytic tasks. We focus on the methods employed in these mechanistic studies using the acyl-transferase family of selected ribozymes as well-studied reference systems. Chemical and biochemical techniques have been used in tandem in order to draw conclusions on the various modes of catalysis employed by the different family members. In turn, this type of mechanistic information may provide a means for the redesign and optimization of existing ribozymes or the basis for new selection systems for more powerful RNA catalysts.     

Purification and characterization of halohydrin hydrogen-halide lyase from a recombinant Escherichia coli containing the gene from a Corynebacterium sp.

Summary An enzyme catalyzing the interconversion of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) was purified from Escherichia coli JM109/ pST001, which carried the gene from Corynebacterium sp. N-1074. The enzyme was crystallized by the addition of ammonium sulphate. The enzyme had a relative molecular mass (M_r) of about 105 000 and consisted of four subunits identical in M_r (approx. 28 000). The enzyme catalysed both the transformation of various halohydrins into the corresponding epoxides with liberation of halide and its reverse reaction. These facts indicated that the enzyme was halohydrin hydrogen-halidelyase. Offprint requests to: T. Nagasawa