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。     

Effects of alternative carbon sources on biological transformation of nitrophenols

The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources. Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol. Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. Throughout the study the hydraulic retention time (HRT) and COD/NO₃ ^-–N ratio were kept as 24 h and 10, respectively. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively. Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate. Nitrate nitrogen removal was always more than 99%. COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7%. The oxidation-reduction potential (ORP) inside the reactors dropped, up to –300 mv, with glucose as carbon source. As the reactors were switched over to methanol, ORP increased to –190 mv. The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run. Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.     

Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45

Microbial metabolism of nitroarenes via o-aminophenols requires the participation of two key enzymes, a nitroreductase and an hydroxylaminobenzene mutase. The broad substrate ranges of the enzymes suggested that they could be used as biocatalysts for the production of substituted o-aminophenols. We have used enzymes from Pseudomonas pseudoalcaligenes JS45 for the conversion of 4-nitrobiphenyl ether to the corresponding o-aminophenol. Partially purified nitrobenzene nitroreductase reduced 4-nitrobiphenyl ether to the corresponding 4-hydroxylaminobiphenyl ether. Partially purified hydroxylaminobenzene mutase stoichiometrically converted the intermediate to 2-amino-5-phenoxyphenol. The results indicate that the enzyme system can be applied for the production of o-aminophenols useful as intermediates for synthesis of commercially important materials. Journal of Industrial Microbiology & Biotechnology (2000) 24, 301–305. Received 13 October 1999/ Accepted in revised form 31 January 2000     

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACT

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

One-step production of picolinic acids from 2-aminophenols catalyzed by 2-aminophenol 1,6-dioxygenase

Picolinic acids have been synthesized previously from catechols by the action of catechol 2,3-dioxygenase and a subsequent chemical reaction in the presence of ammonia. 2-Aminophenol 1,6-dioxygenase catalyzes ring cleavage of several ortho-aminophenols. The ring fission products spontaneously convert to picolinic acids. Resting cells of Escherichia coli DH5α/pNBZ14 harboring the genes for 2-aminophenol 1,6-dioxygenase converted 2-aminophenol and 6-amino-m-cresol to picolinic acid and 5-methylpicolinic acid with yields greater than 90%. The results provide a convenient strategy for the synthesis of substituted picolinic acids from the corresponding aminophenols. Journal of Industrial Microbiology & Biotechnology (2000) 25, 25–28. Received 25 October 1999/ Accepted in revised form 19 April 2000     

On the role of chiral catalysts in the alkenyl zirconocene/zinc addition to aldehydes: a study of ligand loading and asymmetric amplification

Unusual nonlinear asymmetric amplification and chiral ligand loading effects were discovered for the use of catalytic quantities of chiral aminoalcohols in the in situ hydrozirconation-transmetalation-aldehyde addition processes. While the stereochemically most efficient aminothiol ligands demonstrated mechanistically conventional reaction parameters in excellent agreement with Kagan's ML(2) system, the asymmetric induction in the presence of a chiral aminoalcohol was found to vary greatly with loading and %ee of the ligand. Aminothiols remain the ligands of choice for the highly enantioselective formation of allylic alcohols and provide experimentally more predictable reaction variables. However, new, optimized conditions lead to a synthetically useful product %ee using the readily available and scalable aminoalcohol 2a.     

Dimerization of hydroxylated species of m-aminophenol by cytochrome c with hydrogen peroxide

The cytochrome c and hydrogen peroxide-dependent oxidation of m-aminophenol was investigated by electrochemistry and spectrophotometry. The results indicated that the hydroxylated species of m-aminophenol have at least two conjugated substituted groups on the ring system (most possibly, its oxidized form 2-hydroxy-4-iminoquinone), and that the degradation of cytochrome c by hydrogen peroxide can also be prevented in the presence of m-aminophenol. The hydroxyl radical scavengers, mannitol and sodium benzoate, almost completely eliminate the hydroxylation of m-aminophenol. But oxo-heme species scavenger, uric acid, does not inhibit the hydroxylation. Combining the results of mass spectrum, nuclear magnetic resonance and element analysis with that of spectrophotometry, electrochemistry and chemical scavengers, it is suggested that cytochrome c may act as a peroxidase, which facilitates the hydroxylation and subsequent dimerization of m-aminophenol.     

Development of an enzyme-based assay for acetaminophen

A new and novel method for determination of serum acetaminophen is described. The assay, which can be completed in less than 5 min, is based on the enzymatic hydrolysis of acetaminophen, with subsequent colorimetric detection of the aminophenol so produced. Various possible means of aminophenol estimation are described; the final reaction conditions have been optimized for maximum sensitivity and assay speed. This assay compares favorably with other available procedures; it requires only small sample volumes; it is rapid, simple, and highly specific for the parent drug; and it requires neither great technical ability nor expensive instrumentation.