Preparation of 3-deacetyl cephalosporins by Aspergillus niger lipase

Lipase from Aspergillus niger was used for the selective hydrolysis of the 3-O-acetate of cephalosporin C to give an intermediate useful for further chemical elaborations. This lipase was purified to homogeneity and its properties compared with previously published data that present some discrepancies. The lipase proved to be very effective in catalyzing 3-O-acetate hydrolysis and versatile toward substitution on the beta-lactamic ring. In fact, as an example, two other cephalosporinic derivatives, cephalotin and cefotaxime, were efficiently deacetylated. The lipase was immobilized on Eupergit C and employed continuously in either a column or a batch reactor for 2 months without appreciable loss of activity. (c) 1996 John Wiley & Sons, Inc.     

Selective monoacetylation of diol compounds by Aspergillus niger lipase

Summary The primary monoesters of diol compounds were formed exclusively in the reaction with vinyl acetate and Aspergillus niger lipase for 24~72 h. Various diol compounds which included 1,3-butanediol, 1,4-butanediol, 1,5-hexanediol, 1-phenyl-1,2-ethanediol, 1-phenyl-1,3-propanediol, 2, 3, or 4-hydroxybenzyl alcohol, methyl 2, 3-O-acetyl-D-glycopyranosides and phenyl 1-thio--D-xylopyranoside have been examined and showed nearly 100% regioselectivity.     

Studies on the biosynthesis of aspergillin by Aspergillus niger.

Two inhibitors of the biosynthesis of aspergillin, the black spore pigment of Aspergillus niger, have been investigated. 2,4-Dithiopyrimidine exerted its inhibitory effect by intracellularly chelating cupric ion required for normal pigmentation. Dimethylsulfoxide prevented the synthesis of certain phenolic precursors of the native pigment. Partial purification and characterization of pigments from mature cultures revealed the presence of at least three components: (i) a high-molecular-weight (approximately 20,000) native pigment fraction in untreated mold cultures, (ii) a lower-molecular-weight (approximately 5,000) melanin pigment found in both types of inhibited cultures, and (iii) a low-molecular-weight (368) green pigment found only in the 2,4-dithiopyrimidine-inhibited cultures and proposed to be a pentacyclic quinonoid derivative. A pathway for aspergillin biosynthesis is suggested based on these results.     

Glyceride synthesis by a cell-bound lipase from Aspergillus niger

A cell-bound lipase from the mould Aspergillus niger was isolated and purified to homogeneity as judged by disc electrophoresis on a Sephadex G-75 column. Molecular weight determination and electrophoretic mobility (Rf value) indicated that the enzyme differed from that secreted extracellularly. The cell-bound lipase showed a higher capacity than the extracellular enzyme for glyceride synthesis, and reacted in an opposite manner in the synthetic reaction compared with the extracellular enzyme.     

Inulinase biosynthesis using immobilized mycelium of Aspergillus niger

Conidia of Aspergillus niger 20 Osm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 °C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant.     

Strain improvement of Aspergillus niger for enhanced lipase production

The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.     

Effect of maltose on glucoamylase formation by Aspergillus niger

Low levels of glucoamylase are produced when Aspergillus niger is grown on sorbitol, but substitution of the latter by glucose, maltose, or starch results in greater formation of glucoamylase as measured by enzymatic activity. Both glucoamylase I and glucoamylase II are formed in a yeast extract medium; however, glucoamylase I appears to be the only form produced when ammonium chloride is the nitrogen source. Maltose or isomaltose (1.4 x 10(-4)m), but no other disaccharides or monosaccharides, dextrins, dextrans, or starches, stimulated glucoamylase formation when added to mycelia pregrown on sorbitol-ammonium salts. The induction of glucoamylase by maltose was independent of sulfate concentration but showed a dependency on low pH and the absence of utilizable carbon sources.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Construction of Aspergillus niger lipase mutants with oil-water interface independence

Based on previous bioinformational analytical results [Shu ZY, et al. Biotechnol Prog 2009;25:409-16], four A. niger lipase (ANL) mutants, ANL-Ser84Gly, ANL-Asp99Pro, ANL-Lys108Glu and ANL-EαH (obtained by replacing the lid domain of ANL with the corresponding domain from A. niger feruloyl esterase), were constructed to screen out ANL mutants with oil-water interface independence. ANL-S84G displayed a pronounced interfacial activation, while ANL-D99P and ANL-K108E displayed no interfacial activation. The specific activity of ANL-S84G towards p-nitrophenyl esters decreased from 29.8% to 76.5% compared with that of ANL, while the specific activity of ANL-D99P towards p-nitrophenyl palmitate increased 2.2-fold. The thermostability of ANL-K108E was almost unchanged, while the thermostability of ANL-S84G and ANL-D99P significantly decreased compared with that of ANL. The construction of oil-water interface-independent ANL mutants would help to further understand the mechanism of lipase interfacial activation.     

黑曲霉(Aspergillus niger)脂肪酶的纯化及性质分析

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％。对脂肪酶的性质分析表明：该酶分子质量约为35kDa,最适温度和最适pH分别为37℃和9．5,50℃以下和pH6．0～11．0之间保持稳定,属于碱性脂肪酶。Mg^2＋、Ca^2＋、Cu^2＋、Zn^2＋、Co^2＋、Mn2^＋对该酶有激活作用,而Al^3＋、Fe^2＋、Fe^3＋对酶有严重抑制作用。变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活。用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性。外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期。在一定质量比条件下,该酶有良好的抗蛋白酶性质。     

Production and partial characterisation of extracellular lipase from Aspergillus niger

Summary The production and certain kinetic characteristics of extracellular lipase from Aspergillus niger were investigated. It was possible to substantially enhance the activity of excreted lipase by optimising the interaction between carbon and nitrogen sources applying a two-parameter complete experimental design and response surface analysis. The enzyme was partially purified and a number of kinetic characteristics such as optimum pH and temperature, thermal and pH stability and K_m were determined and discussed. The elevated levels of lipase activity (40.5 U/ml) found in this work competed favourably with most of those reported for lipase hyperproducing fungi.     

Purification of lipase from Aspergillus niger and its characterization analysis

从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50％.对脂肪酶的性质分析表明:该酶分子质量约为35 kDa,最适温度和最适pH分别为37℃和9.5,50℃以下和pH6.0～11.0之间保持稳定,属于碱性脂肪酶.Mg2+、Ca2+、Cu2+、Zn2+、Co2+、Mn2+对该酶有激活作用,而Al3+、Fe2+、Fe3+对酶有严重抑制作用.变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活.用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性.外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期.在一定质量比条件下,该酶有良好的抗蛋白酶性质.     

Glucose oxidase biosynthesis using immobilized mycelium of Aspergillus niger

A simple method is described for the immobilization of Aspergillus niger GIV-10 which produces an extracellular glucose oxidase. A. niger conidia were immobilized on sintered glass Raschig rings, pumice stones or polyurethane foam. Mycella growing out from the spores produced extracellular glucose oxidase: the highest production was with the pumice stone carrlers. This technique facilitates the growth of the filamentous cultures in the spongy structure of a support with continuous accumulation of biomass. After 24 to 36 h, a culture liquid with 2.7 to 3.1 U of glucose oxidase/ml was obtained. This procedure also made possible repeated batch enzyme production and as many as 25 subsequent 24-h batches could be fermented by using the same carrier with only a small loss of glucose oxidase activity.The authors are with the Institute of Microbiology, M. Curie-Sklodowska University, Akademicka 19, 20-003 Lublin, Poland.     

不依赖油水界面激活的黑曲霉脂肪酶突变体的构建

为获得不依赖油水界面激活的黑曲霉脂肪酶 (ANL) 突变体,在生物信息学分析基础上,对黑曲霉脂肪酶盖子结构域两侧铰链区的氨基酸残基进行了置换突变,获得两个黑曲霉脂肪酶突变体 (ANL-Ser84Gly和ANL-Asp99Pro)。对不同浓度对硝基苯丁酸酯的水解活性检测结果表明：ANL-Ser84Gly的催化活性仍依赖油水界面,而ANL-Asp99Pro的催化活性不再依赖油水界面。底物特异性检测结果表明：较ANL而言,ANL-Ser84Gly的比活力显著降低,其水解对硝基苯棕榈酸酯、对硝基苯豆蔻酸酯、对硝基