Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

2-Alkyl(aryl)-quinazolin-4(3H)-thiones, 2-R-(quinazolin-4(3H)-ylthio)carboxylic acids and amides: synthesis,molecular docking,antimicrobial and anticancer properties

In this study, a series of novel 2-alkyl(aryl)-quinazolin-4(3H)-thiones, 2-R-(quinazolin-4(3H)-ylthio)carboxylic acids and amides were synthesized and evaluated for antimicrobial and anticancer activities. Their structure was confirmed by elemental analysis and spectral data (FT-IR, LC-MS, ¹H-NMR). Antimicrobial activity was tested in vitro against Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Candida albicans and NCI in vitro preliminary anticancer activity against nine different cancer types. The most active antibacterial and antifungal compounds were: 2.1, 2.2 and 2.4. The introduction of the carboxylic acid or amide residue into the fourth position of quinazolin-4(3H)-thione resulted in the absence of antimicrobial activity. Substance 3.8 inhibited renal cancer UO-31 line and 2.18 – leukemia CCRF-CEM. The results of in silico molecular docking for DHFR and CK2 kinase had no correlation with in vitro properties, proposing the presence of other biological activity pathways.     

哈氏弧菌脂酰-ACP合成酶的体外功能

【目的】系统鉴定哈氏弧菌脂酰-ACP合成酶(Acyl-ACP synthetase,Aas S)以不同链长游离脂肪酸和非脂肪链羧酸作为底物的体外催化反应。【方法】利用非变性蛋白质凝胶电泳和紫外分光光度计法从定性和定量两个方面分析了Aas S的体外催化功能与活性。【结果】Aas S能够催化不同链长直链的自由脂肪酸合成脂酰-ACP,其中以C6–C12作为底物时活性最高;以羟基脂肪酸作为底物的情况下,Aas S催化C8–C14的羟基脂肪酸有较高的活性。非脂肪链羧酸类作为底物的反应中,20种蛋白质氨基酸、苯甲酸和水杨酸均可以作为Aas S的底物,合成相应的脂酰-ACP。【结论】本研究系统地证明了哈氏弧菌脂酰-ACP合成酶(Aas S)对不同底物的不同催化活性,为生物体内氨基酸代谢和菌黄素合成代谢的研究提供了可行性的分析依据。     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.     

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

Abstract

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe₃O₄), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe₃O₄ nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60?°C, free lipase lost its initial activity within 80?min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120?min of heat treatment at 60?°C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

盐水球菌Salinicoccus ventosaetal B2-3-5的代谢产物分析及其抑制酪氨酸酶活性的机制

[背景] 酪氨酸酶是黑色素合成过程中的关键酶,也是引起人体色素障碍性疾病和产生果蔬酶促褐变的主要原因。目前,酪氨酸酶抑制剂的开发已引起广泛关注,但一些酪氨酸酶抑制剂如熊果苷、曲酸等均存在一定的安全隐患。微生物资源丰富且具有许多优点,从微生物中寻找特异性强、高效的酪氨酸酶抑制剂已成为该领域研究的热点。[目的] 通过测定分离自新疆乌鲁木齐达坂城盐湖的盐水球菌Salinicoccus ventosaetal B2-3-5和B6-1-4代谢物提取物对酪氨酸酶活性的影响,比较2株菌发酵过程中代谢物的差异,了解所筛选菌株B2-3-5抑制酪氨酸酶活性的机制。[方法] 以曲酸为阳性对照分别测定B2-3-5和B6-1-4这2个菌株发酵产生的代谢物提取物对蘑菇酪氨酸酶的抑制活性;应用LC-MS代谢组学方法检测2株菌在相同条件下产生的所有代谢物质;采用单变量、多元变量、正交偏最小二乘判别分析（Orthogonal Partial Least Squares-Discrimination Analysis,OPLS-DA）法识别差异代谢物;利用层次聚类分析（Hierarchial Cluster Analysis,HCA）法对识别的差异物进行聚类分析;通过Kyoto Encyclopedia of Genes and Genomes （KEGG）代谢通路对比法分析这些差异代谢物主要参与的代谢途径。[结果] 菌株B2-3-5代谢物提取物对蘑菇酪氨酸酶二酚酶活性的抑制率为67%,其IC₅₀为0.277 mg/mL,同属菌株B6-1-4代谢物提取物则对酪氨酸酶无抑制活性。采用代谢组学的检测方法从2株菌的代谢物中筛选出63个差异代谢物,其中氨基酸类化合物、维生素类化合物和羧酸类化合物的种类及相对含量均是B2-3-5菌株明显高于B6-1-4菌株。通过代谢途径分析发现这些差异代谢物主要参与15个代谢通路,其中维生素B6生物合成通路的影响较为显著。[结论] 推测B2-3-5菌株可能是通过增加一些氨基酸类、维生素类及羧酸类等小分子化合物的含量来抑制酪氨酸酶活性。维生素B6代谢途径的上调也表明菌体细胞可通过产生维生素B6与酪氨酸酶中的必需氨基作用或清除酶催化循环过程中产生的活性氧自由基（reactive oxygen species,ROS）来抑制酪氨酸酶活性。     

Novel Sesquiterpene Alcohol Prepared from the Oxidation of α-Longipinene with Lead Tetraacetate

The aromatic side chain of cationomycin (1) was modified by the coupling the sodium salt of deacylcationomycin (2) with several aromatic carboxylic acids. There was a linear relationship between the logarithmic reciprocal minimal inhibitory concentration of the derivatives against Bacillus subtilis IFO 3513 and the logarithmic transport efficiency for the sodium ion. The anticoccidial activity of the anisyl analog (3) was higher than that of the parent compound (1).     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

Synthesis and biological evaluation of O-methyl and O-ethyl NSAID hydroxamic acids

This paper reports the synthesis of O-methyl and O-ethyl NSAID hydroxamic acids, their antimicrobial activities, and their ability to inhibit urease and soybean lipoxygenase activities. Ibuprofen and fenoprofen hydroxamic acids with free hydroxy groups present the highest antimicrobial activity, while indomethacin and diclofenac analogs show significantly lower antimicrobial activity. Diclofenac hydroxamic acid 4e exerts the highest anti-urease activity. Indomethacin O-ethyl hydroxamic acid 3h and ibuprofen O-benzyl hydroxamic acid 4b exert significant inhibitory activities on soybean lipoxygenase. Fenoprofen and indomethacin O-ethyl hydroxamic acids 3b and 3h and diclofenac and indomethacin O-benzyl analogs 4g and 4i highly inhibit lipid peroxidation. The highest antioxidant activity was shown by fenoprofen derivative 3b.     

SYNTHESIS,HYDROLYSIS AND ANTI-EBV ACTIVITY OF A SERIES OF 3′-MODIFIED cycloSal-BVDUMP PRONUCLEOTIDES

A series of cycloSal-BVDUMP phosphate triesters has been prepared. The prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a series of 3′-O-acyl-modified derivatives having carboxylic acids with different lipophilicity or a L-configurated α-amino acid (phenylalanine) was prepared. The hydrolysis properties in phosphate buffer PBS as well as in PBS containing pig liver esterase (PLE) will be described. Finally, the biological activity against EBV has been determined.     

TADH,the thermostable alcohol dehydrogenase from <Emphasis Type="Italic">Thermus</Emphasis> sp. ATN1: a versatile new biocatalyst for organic synthesis

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.     

Chemical modification of lactase for immobilization on carboxylic acid-functionalized microspheres

Abstract

Surface interactions between an enzyme and support influence the retention of activity after immobilization. Chemical modification of enzymes prior to immobilization may be used to alter these interactions and enhance activity retention. Lactase (A. oryzae) was covalently conjugated to P(S/V-COOH) microspheres, with surface carboxylic acid densities of 9 μeq/g and 137 μeq/g, using carbodiimide chemistry. Under optimum pH and temperature conditions, activity retention was greater when the enzyme was conjugated to microspheres containing a lower density of surface carboxylic acid groups (32% activity retention) than when the enzyme was conjugated to microspheres having a greater density of surface carboxylic acid groups (11% activity retention). Chemical modification of lactase carboxylic acid groups with glucosamine prior to immobilization was evaluated as a means to increase activity retention. Under optimal conditions, modification resulted in a 17% decrease in soluble enzyme activity compared to the native enzyme. However, immobilization of the modified enzyme yielded 85% and 64% activity retention after conjugation to microspheres with a lower and higher density of surface carboxylic acid groups, respectively. The results suggest that increases in surface carboxylic acid density on the carrier promote the loss of lactase activity after immobilization, and chemical modification of the enzyme with glucosamine provides a means to retain catalytic activity after attachment to these supports.     

Cloning and characterization of a novel thermostable amidase,Xam, from Xinfangfangia sp. DLY26

Objective

Identification and characterization of a novel thermostable amidase (Xam) with wide pH tolerance and broad-spectrum substrate specificity.

Results

Xam was identified from non-thermophilic Xinfangfangia sp. DLY26 and its acyl transfer activity was investigated. Recombinant Xam was optimally active at 60 °C and pH 9.0. The enzyme had a half life of 18 h at 55 °C and maintained more than 60?% of its maximum activity in the range of pH 3.0–11.0. Additionally, Xam exhibited broad substrate specificity towards aliphatic, aromatic, and heterocyclic amides.

Conclusions

These unique properties make Xam a promising biocatalyst for production of important hydroxamic acids at elevated temperatures.

     

具有优良抑菌特性乳酸菌的筛选鉴定及活性物质检测

【背景】有益性乳酸菌在人体和动物体内分布极为广泛,是维持胃肠道菌群平衡、提高机体免疫力的主力军。近年来,为了解决禁用抗生素而导致动物发病率不断增高的问题,分析和研究乳酸菌及其所产活性物质的益生特性并开发新型饲料添加剂成为一个重要手段。【目的】本实验旨在从土壤中分离筛选出具有优良抑菌特性的乳酸菌,并对其所产活性物质的特性进行分析评价。【方法】采用溴甲酚紫平板法筛选并结合抑菌能力检测,得到2株具有优良抑菌特性的产酸菌株,分别命名为H-3和H-4。经形态学鉴定及16S rRNA基因序列测定后,对2株菌分别进行生长曲线和产酸量检测;通过排除酸处理、蛋白酶处理和热处理的方法分析2株菌所产抑菌物质的有效成分。【结果】H-3和H-4菌株经初步鉴定为乳酸片球菌(Pediococcus acidilactici),2株菌均具有良好的生长性能及产酸性能。菌株发酵上清液对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、猪霍乱沙门氏菌(Salmonella choleraesuis)、福氏志贺氏菌(Shigella flexneri)均表现出明显的抑...     

Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50?mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50?°C and 50?mM, respectively. The enzyme was quite stable for more than 6?h at 30?°C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50?mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120?min, respectively, using 5?mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383?mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.     

施用韩国假单胞菌(Pseudomonas koreensis)CLP-7对连作烟田土壤质量及微生物群落功能多样性的影响

针对目前连作导至植烟土壤酸化和根茎病害发生重的问题,田间条件下,研究了嗜酸性韩国假单胞Pseudomonas koreensis CLP-7对酸化植烟土壤pH、养分、酶活性及微生物群落功能多样性的影响。结果表明:CLP-7可以提高酸化植烟土壤pH;同时,CLP-7显著提高根际土壤速效钾、有效磷、铵态氮、硝态氮和有机质含量,其中有机质含量增加最显著;根际土壤脲酶、蔗糖酶活性呈上升趋势且均高于未施菌土壤,过氧化氢酶活性变化不明显。CLP-7处理不同时间的土壤微生物代谢多样性指数表现出明显的差异,施菌30 d时Shannon指数、Simpson指数,Richness及McIntosh指数均达到最高;所有处理的Pielou指数变化较小。微生物碳代谢主成分分析结果显示,施菌30 d时,根际土壤微生物对碳源利用与其他处理差异显著,主要为羧酸类和糖类碳源;随着施菌时间增加,微生物对氨基酸类、羧酸类、双亲化合物类、聚合物类和糖类利用率明显提高,说明CLP-7有利于提高连作烟田根际土壤微生物对碳源的利用能力。综上所述,P.koreensis CLP-7能够明显提高土壤pH、土壤酶活性,增加土壤养分含量和土壤微生物群落功能多样性,进而改善酸化植烟土壤质量,在微生物修复酸化土壤和减轻根茎病害发生的烟草绿色防控中具有较大的应用潜力。     

Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol™), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37°C, respectively. The half-life of enzyme activity at 37°C was 192 h and 90% of its activity remained after 3 h incubation at 40°C. Divalent metals was found to inhibit the activity of enzyme. The K _m values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C₁∼C₄ and C₁₀) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.     

Mechanism of inhibition of cytoplasmic streaming by myrmicacin (Β-hydroxydecanoic acid) inChara andSpirogyra

Summary Myrmicacin (-hydroxydecanoic acid) applied externally inhibited the cytoplasmic streaming ofChara cells in acidic (pH 4.5) but not in neutral (pH 7.5) solution. Application by intracellular perfusion at neutral pH also did not inhibit the streaming. Other carboxylic acids could inhibit the streaming when given as an acidic external solution. Inhibition of the cytoplasmic streaming was concluded to occur due to acidification of the intracellular pH upon application of an acidic external medium of these carboxylic acids. The cytoplasmic streaming ofSpirogyra was also inhibited by myrmicacin and acetic acid in acidic solution.Abbreviations APW artificial pond water - CB cytochalasin B - DMGA 3.3-dimethylglutaric acid - DMSO dimethylsulfoxide - EGTA ethyleneglycol-bis-(\-aminoethyl ether)N - N tetraacetic acid - HEPES 2-hydroxyethylpiperazine - N ethanesulfonic acid - MES 2-(N-morpholino)ethanesulfonic acid - pH _i intracellular pH - Tris trishydroxyaminomethane