Enzymatic synthesis and properties of uridine-5'-O-(2-thiodiphospho)-N-acetylglucosamine

This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.     

Enzymatic synthesis of the acrylic esters: a comparative study

Various acrylic esters were synthesized by Candida cylindracea lipase (CCL) catalysed transesterification with different alcohols. A comparative study was carried out using 2,3-butanedione mono-oxime acrylate and vinyl acrylate as acylating agents. The rate of conversion was faster when oxime acrylate was used as acylating agent as compared to vinyl acrylate. Effect of solvents on the rate of conversion was studied and diisopropyl ether (DIPE) was proved to be a better solvent as compared to CHCl₃ and THF. The effect of various structural aspects of the substrates on the rate of conversion was studied. Among the linear alcohols studied, ease of conversion was found to be in the order of n-octanol>n-hexanol>n-butanol. Up to 80% conversion was achieved in the case of cyclohexyl methanol.     

Enzymatic synthesis of UDP-galactose on a gram scale

Uridine 5′-diphospho-- -galactose (UDP-Gal) was synthesized on a gram scale from uridine 5′-diphospho-- -glucose and - -galactose 1-phosphate using the enzymes galactose-1-phosphate uridyltransferase (EC 2.7.7.12), phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.27). The synthesis was performed in a repetitive batch mode in which the enzymes, some of which are expensive, were used in 16 subsequent batches without any loss of enzyme activity. The space time yield of the synthesis was 7.1 g/l d. The overall yield of the synthesis amounted to 40% and 1.1 gram of pure UDP-Gal was obtained.     

Enzymatic synthesis of a 6-sialyl lactose analogue using a pH-responsive water-soluble polymer support

The Letter describes a strategy for the enzymatic synthesis of glycans based on a pH-responsive water-soluble polymer. In neutral condition, the polymer is water-soluble and convenient for in-solution enzymatic synthesis, whereas in acidic condition (pH lower than 4.0), the polymer disconnects with the product and becomes insoluble, which can be easily removed. A 6-Sialyl lactose analogue was synthesized as a model reaction using this approach.     

Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli

A gene corresponding to a maltogenic amylase (MAase) in Lactobacillus gasseri ATCC 33323 (lgma) was cloned and expressed in Escherichia coli. The recombinant LGMA was efficiently purified 24.3-fold by one-step Ni-NTA affinity chromatography. The final yield and specific activity of the purified recombinant LGMA were 68% and 58.7 U/mg, respectively. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 55 degrees C and pH 5. The relative hydrolytic activities of LGMA to beta-CD, soluble starch or pullulan was 8:1:1.9. The activity of LGMA was strongly inhibited by most metal ions, especially Zn(2+), Fe(2+), Co(2+) and by EDTA. LGMA possessed some unusual properties distinguishable from typical MAases, such as being in a tetrameric form, having hydrolyzing activity towards the alpha-(1,6)-glycosidic linkage and being inhibited by acarbose.     

Enzymatic synthesis of derivatives of vitamin A in organic media

The present article provides an enzymatic method of retinol esterification to reduce photodestruction and irritation problems characteristic of retinol. More specifically, it relates to a method of synthesising retinyl adipate, retinyl succinate, retinyl oleate and retinyl lactate greatly appreciated by cosmetic manufacturer. Desired compounds can be synthesised directly using Candida antarctica lipase and Rhizomucor miehei lipase in organic media.     

细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达

【目的】实现地衣芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的高效异源表达,并研究该重组酶的酶学性质。【方法】克隆巨大芽孢杆菌木糖异构酶基因的启动子区域及其调控蛋白,构建一个大肠杆菌/芽孢杆菌穿梭型诱导表达质粒,使用该诱导型启动子介导麦芽糖淀粉酶编码基因,实现其在枯草芽孢杆菌中的功能表达。对重组枯草芽孢杆菌的诱导条件进行优化,提高麦芽糖淀粉酶的产量。【结果】获得了诱导表达麦芽糖淀粉酶基因的重组枯草芽孢杆菌菌株。最适诱导温度为45°C,最适诱导剂添加浓度为1%,最适添加诱导剂时间为接种培养9 h后。重组酶蛋白分子量大小为67 k D,对该酶的酶学性质研究发现,以可溶性淀粉为底物,反应生成麦芽糖和葡萄糖,其中麦芽糖含量为60.42%。重组酶最适作用温度为45°C,最适作用p H为6.5,Ca2+、Co2+、EDTA对该重组麦芽糖淀粉酶具有激活作用。【结论】通过木糖诱导表达系统可以实现麦芽糖淀粉酶在枯草芽孢杆菌中的高效诱导型表达,酶活最高可达296.64 U/m L发酵液,在工业上有着较好的应用前景。     

Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus

Abstract We have cloned and expressed a novel maltogenic alpha-amylase from B. stearothermophilus on plasmid in B. subtilis . Originally the plasmid was very unstable in the absence of selection, but was stabilized due to a spontaneous, copy number reducing mutation. The promoter region and the extension of the gene have been analysed, and a provisional DNA sequence has been determined. The N-terminal of the mature amylase has been determined and shown to be in accordance with signal peptidase processing after a typical Gram-positive signal sequence of 33 amino acids.     

Enzymatic synthesis of trehalose esters having lipophilicity

To improve trehalose lipophilicity, trehalose was regioselectively esterified with vinyl fatty acid esters in dimethyl formamide by protease from Bacillus subtilis to give 6-O-lauroyltrehalose, 6-O-myristoyltrehalose, 6-O-palmitoyltrehalose, 6-O-stearoyltrehalose, 6-O-oleoyltrehalose and 6-O-linoleoyltrehalose. The influence of structural variation by changing fatty acid substitute was examined by measurement of the surface tension and biodegradability.     

Enzymatic synthesis of a novel glycolipid biosurfactant, mannosylerythritol lipid-D and its aqueous phase behavior

Mannosylerythritol lipids (MELs) produced by yeasts are one of the most promising glycolipid biosurfactants. In this study, we succeeded in the preparation of a novel MEL homolog having no acetyl groups, namely MEL-D. MEL-D was synthesized by lipase-catalyzed hydrolysis of acetyl groups from a known MEL, and identified as 4-O-[2′,3′-di-O-alka(e)noyl-β-d-mannopyranosyl]-(2R,3S)-erythritol. The obtained MEL-D showed a higher critical aggregation concentration (CAC = 1.2 × 10⁻⁵ M) and hydrophilicity compared to known MELs, retaining an excellent surface tension lowering activity (the surface tension at the CAC was 24.5 mN/m). In addition, we estimated the binary phase diagram of the MEL-D–water system based on a combination of visual inspection, polarized optical microscopy, and SAXS measurement. From these results, MEL-D was found to self-assemble into a lamellar (L_α) structure over all ranges of concentration. Meanwhile, the one-phase L_α region of MEL-D was extended wider than those of known MELs. MEL-D might keep more water between the polar layers in accordance with the extension of the interlayer spacing (d). These results suggest that the newly obtained MEL-D would facilitate the application of MELs in various fields as a lamellar-forming glycolipid with higher hydrate ability.     

MALDI-TOF Mass Spectrometry as a Powerful Tool to Study Enzymatic Processing of DNA Lesions Inserted into Oligonucleotides

Abstract

MALDI-TOF mass spectrometry measurements, coupled with either exonuclease or DNA N-glycosylases digestions of lesion-containing oligonucleotides, were used to assess biochemical features of several oxidative DNA damage. The latter analytical approach was shown to be an informative and efficient alternative technique to conventional electrophoresis and chromatographic analyses.     

Enzymatic synthesis of heparin related polysaccharides on sensor chips: rapid screening of heparin-protein interactions

The biological roles of heparin (HP) and heparan sulfate (HS) are mediated mainly through their interaction with proteins. In the present work, we provide a rapid method for screening HP/HS-protein interactions providing structural data on the key sulfo groups that participate in the binding. A library of polysaccharides structurally related to HP was prepared by immobilizing the biotinylated N-sulfated K5 polysaccharide (N-sulfoheparosan) on sensor chips followed by selective modification of this polysaccharide with enzymes that participate in HP/HS biosynthesis. The polysaccharides synthesized on the surface of the sensor chips differ in the number and position of sulfo groups present both on uronic acid and glucosamine residues. Surface plasmon resonance was used to measure the interaction of each member of this polysaccharide library with antithrombin III (ATIII), to afford structural information on sulfo groups required for this HP/HS-protein interaction. This method is viewed as widely applicable for the study of the structure-activity relationship (SAR) of HP/HS-protein interactions.     

Enzymatic synthesis of an acylated phloroglucinol derivative with phloroglucinol and vinyl octanoate in acetonitrile

Monooctanoyl phloroglucinol with anti-microbial activities was synthesized from phloroglucinol (50 mM) and vinyl octanoate (150 mM) with agitation at 40 °C using 10 mg lipase AK (Pseudomonas fluorescens) in acetonitrile (1 ml, aw=0.07). The yield was approximately 70% as conversion rate of phloroglucinol over 3 days. The product, at 205 g ml^–1, was bacteriocidal against Escherichia coli and Staphylococcus epidermidis.     

Enzymatic synthesis of a series of alkyl esters using novozyme 435 in a packed-bed,miniaturized, continuous flow reactor

Using lipase catalysed, enzymatic esterification as a model reaction, we successfully demonstrate the use of miniaturized technology for biocatalytic reactions. Benchmarked against batch reactions, nine alkyl esters have been synthesized effectively, using Novozyme 435 in a pressure driven, packed-bed, miniaturized, continuous flow reactor. In some cases close to 100% ester conversions were obtained. The paper also demonstrates the ability to screen the enzyme for substrate specificity.     

Enzymatic synthesis of oligosaccharides from branched cyclodextrins

6'-alpha-Maltosyl-maltotriose and 6'-alpha-D-glucosyl-maltotriose were prepared from Novamyl degradation of 6-O-alpha-maltosyl-alpha-cyclodextrin and 6-O-alpha-D-glucosyl-alpha-cyclodextrin, respectively. NMR spectroscopy was used to elucidate their structural identities, in a combination of COSY experiments. Further, a mechanism for the degradation was proposed based on the Novamyl active site geometry.     

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase

A novel tyrosinase inhibitor, an arbutin derivative having undecylenic acid at the 6-position of its glucose moiety, was enzymatically synthesized. Its inhibitory activity was studied in vitro by using catechol and phenol as substrates. The IC₅₀ value of the arbutin ester on tyrosinase using catechol (4 × 10⁻⁴ M) was 1% of that when arbutin (4 × 10⁻² M) was used. Using phenol, IC₅₀ of the arbutin ester (3 × 10⁻⁴ M) as substrate was 10% of that of arbutin (3 × 10⁻³ M). These results suggest that the arbutin ester inhibits the latter part of the tyrosinase reaction, which consists of hydroxylation and oxidation.     

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with ¹³C-¹H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all ¹H and ¹³C chemical shifts is presented.     

Enzymatic synthesis of the Tn antigen

The enzymatic synthesis of the Tn antigen (GalNAc-α-O-Ser), a glyco-aminoacid of great biological importance, is reported. The reaction was promoted by commercial α-N-acetylgalactosaminidase from Acremonium sp., using p-nitrophenyl-α-N-acetylgalactosamine as the donor. The kinetics were monitored by capillary electrophoresis and LC–UV-MS. For unprotected serine, the role of pH and temperature was investigated, finding that pH 5 and T = 18 °C gave the best yield. Under these conditions a significant increase of the reaction rate was observed in comparison with previous literature data, using unprotected serine. The role of the bulkiness of the serine protecting groups on the yield was additionally considered, as well as the kinetic profiles generated by the use of two differently protected aminoacids. By proper choice of the protecting group, the reaction yield then increased from 5% (with unprotected serine) to about 50% (with N-Boc and N-methoxycarbonyl serine).     

酶法合成天冬甜精中的几个问题

本文从酶、酶的作用底物和合成体系三个方面综述了酶法合成天冬甜精中的几个问题。     

Two-step enzymatic synthesis of UDP-N-acetylgalactosamine

UDP-GalNAc has been synthesised with high yield from GalNAc, UTP and ATP using recombinant human GalNAc kinase GK2 and UDP-GalNAc pyrophosphorylase AGX1. Both enzymes have been prepared in one step from 1 L cultures of transformed Escherichia coli and the UDP-GalNAc produced has been purified by a simple procedure. The method described is a rapid and efficient means to produce UDP-GalNAc as well as analogues like UDP-N-azidoacetylgalactosamine (UDP-GalNAz).