RESUMO
The Morita-Baylis-Hillman (MBH) reaction affords highly functionalised allylic alcohols containing a new stereogenic centre. These MBH adducts are very versatile and have been transformed into a large range of products, some of which have medicinal potential. Several examples of asymmetric syntheses of MBH adducts have been reported, although a generally applicable method remains to be developed. Biocatalytic approaches for the synthesis and enzymatic kinetic resolution of MBH adducts have been reported, and are discussed in detail in this review. Enzymes able to catalyse the asymmetric MBH reaction have been identified, but selectivity and efficiency have generally been low. Lipases, esterases and nitrile-converting enzymes have all been successfully applied in the resolution of MBH adducts, with excellent selectivity being realised in most cases.
Assuntos
Biocatálise , CatáliseRESUMO
This tutorial review focuses on recent advances in technologies for enzyme immobilisation, enabling their cost-effective use in the bio-based economy and continuous processing in general. The application of enzymes, particularly in aqueous media, is generally on a single use, throw-away basis which is neither cost-effective nor compatible with a circular economy concept. This shortcoming can be overcome by immobilising the enzyme as an insoluble recyclable solid, that is as a heterogeneous catalyst.
Assuntos
Biotecnologia/economia , Enzimas Imobilizadas/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Reutilização de Equipamento , Grafite/química , Lipase/química , Lipase/metabolismo , Magnetismo , Estruturas Metalorgânicas/químicaRESUMO
BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.
Assuntos
Genoma Bacteriano/genética , Anotação de Sequência Molecular , Rhodococcus/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos/genética , Farmacorresistência Bacteriana/genética , Nitrilas/metabolismo , Oxirredutases/genética , Rhodococcus/metabolismoRESUMO
The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8â¯M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80â¯mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65⯰C. At these conditions, the enzyme exhibited an activity of 0.44⯱â¯0.03⯵Mâ¯min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25⯰C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.
Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Oxirredutases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Escherichia coli , Interações Hidrofóbicas e Hidrofílicas , Corpos de Inclusão , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Solubilidade , Espectrometria de Massas em TandemRESUMO
The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to large (325 Da) were evaluated. Larger compounds included those with a bi-aryl axis, prepared by the Suzuki coupling reaction, Morita-Baylis-Hillman adducts, heteroatom-linked diarylpyridines prepared by Buchwald-Hartwig cross-coupling reactions and imidazo[1,2-a]pyridines prepared by the Groebke-Blackburn-Bienaymé multicomponent reaction. The enzyme active site was moderately accommodating, accepting almost all of the small aromatic nitriles, the diarylpyridines and most of the bi-aryl compounds and Morita-Baylis-Hillman products but not the Groebke-Blackburn-Bienaymé products. Nitrile conversion was influenced by steric hindrance around the cyano group, the presence of electron donating groups (e.g., methoxy) on the aromatic ring, and the overall size of the compound.
Assuntos
Cobalto/química , Hidroliases/química , Rhodococcus/enzimologia , Catálise , Hidroliases/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Especificidade por SubstratoRESUMO
Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.
Assuntos
Cobalto/metabolismo , Hidroliases/metabolismo , Ferro/metabolismo , Burkholderiaceae/metabolismo , Catálise , Escherichia coli/metabolismo , Metaloproteínas/metabolismo , Methylobacteriaceae/metabolismo , Pseudomonas/metabolismoRESUMO
Commercially, nitrilases are valuable biocatalysts capable of converting a diverse range of nitriles to carboxylic acids for the greener synthesis of chemicals and pharmaceuticals. Nitrilases are widespread in nature and are both important components of metabolic pathways and a response to environmental factors such as natural or manmade nitriles. Nitrilases are often grouped together on a genome in specific gene clusters that reflect these metabolic functions. Although nitrilase induction systems are still poorly understood, it is known that a powerful Rhodococcal transcription regulator system permits accumulation of intracellular nitrilase of up to 30-40% of total soluble protein in wild type Rhodococcous rhodochrous and host Streptomyces strains. Nitrilase expression inducer molecules encompass a broad range of aliphatic, aromatic and heteroaromatic nitriles, as well as some secondary and tertiary amides that are resistant to nitrilase degradation.
Assuntos
Aminoidrolases/biossíntese , Aminoidrolases/genética , Bactérias/enzimologia , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Biocatálise , Indução Enzimática , Família Multigênica , Rhodococcus/enzimologia , Rhodococcus/genética , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por SubstratoRESUMO
"Dirty" glycerol from biodiesel production is having a considerable environmental impact since its disposal is expensive and difficult. The increased biodiesel production in the last two decades has forced glycerol prices down, thereby making it now unprofitable for chemical companies to produce. The problem lies with the impurities of the biodiesel conversion process usually ending up within the crude glycerol fraction. These impurities are often too costly to purify with current processes, particularly for small scale producers. A wide variety of industries, including the paint, tobacco, food and pharmaceutical industries, utilize glycerol as part of their technology or products. However, the crude glycerol from biodiesel production is not of a high enough grade to be used in these industries. Biodiesel-produced crude glycerol is therefore cheap, readily available and presents itself as an attractive carbon source for industrial microbial production systems synthesizing value-added products. This mini-review will look at (a) microbial production processes which use crude glycerol to produce high-value products (product-driven research) and (b) genetic engineering of microbes which is aimed at improving microbial "dirty" glycerol utilization (substrate driven research).
Assuntos
Biocombustíveis , Fermentação , Glicerol/metabolismo , Microbiologia Industrial , Bactérias/genética , Bactérias/metabolismo , Carbono , Engenharia GenéticaRESUMO
The acylation of the acetonitrile anion with lactones and esters in ethereal solvents was successfully exploited using inexpensive KOt-Bu to obtain a variety of ß-ketonitriles and trifunctionalized building blocks, including useful O-unprotected diols. It was discovered that lactones react to produce the corresponding derivatized cyclic hemiketals. Furthermore, the addition of a catalytic amount of isopropanol, or 18-crown-6, was necessary to facilitate the reaction and to reduce side-product formation under ambient conditions.
RESUMO
Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-L-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.
Assuntos
Glicerol/química , Polilisina/biossíntese , Cloreto de Sódio/análise , Streptomyces/metabolismo , Diamino Aminoácidos/metabolismo , Biocombustíveis/análise , Biomassa , Carboidratos/análise , Biologia Computacional , Meios de Cultura/química , Fermentação , Perfilação da Expressão Gênica , Cloreto de Potássio/análise , Análise de Sequência de RNARESUMO
Suberase®, a commercial laccase from Novozymes, was used to catalyse the synthesis of coumestans. The yields, in some cases, were similar to or better than that obtained by other enzymatic, chemical or electrochemical syntheses. The compounds were screened against renal TK10, melanoma UACC62 and breast MCF7 cancer cell-lines and the GI50, TGI and LC50 values determined. Anticancer screening showed that the cytostatic effects of the coumestans were most effective against the melanoma UACC62 and breast MCF7 cancer cell-lines exhibiting potent activities, GI50=5.35 and 7.96µM respectively. Moderate activity was obtained against the renal TK10 cancer cell-line. The total growth inhibition, based on the TGI values, of several of the compounds was better than that of etoposide against the melanoma UACC62 and the breast MCF7 cancer cell lines. Several compounds, based on the LC50 values, were also more lethal than etoposide against the same cancer cell lines. The SAR for the coumestans is similar against the melanoma UACC62 and breast MCF7 cell lines. The compound having potent activity against both breast MCF7 and melanoma UACC62 cell lines has a methyl group on the benzene ring (ring A) as well as on the catechol ring (ring B). Anticancer activity decreases when methoxy and halogen substituents are inserted on rings A and B.
Assuntos
Antineoplásicos/farmacologia , Cumarínicos/farmacologia , Lacase/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Biocatálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/química , Cumarínicos/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular ß-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.
Assuntos
Microbioma Gastrointestinal/genética , Glicosídeo Hidrolases/genética , Isópteros/microbiologia , Metagenômica , Animais , Celulases/química , Celulases/classificação , Celulases/genética , Celulases/isolamento & purificação , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/isolamento & purificação , Hidrólise , Isópteros/enzimologia , Isópteros/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Simbiose/genéticaRESUMO
A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes. The amino acid sequence lengths of the seven FAE encoding open reading frames (ORFs) ranged from 260 to 274 aa and encoded polypeptides of between 28.9 and 31.4 kDa. The highest sequence identity scores for the seven ORFs against the GenBank database were between 45 and 59 % to a number of carboxyl ester hydrolyses. The seven FAE primary structures contained sequence motifs that correspond well with a classical pentapeptide (G-x-S-x-G) serine hydrolyse signature motif which harbours the catalytic serine residue in other FAE families. Six of the seven fae genes were successfully expressed heterologously in Escherichia coli, and the purified enzymes exhibited temperature optima range of 40-70 °C and the pH optima of between 6.5 and 8.0. The k(cat)/K(M) ratios for the six characterised FAEs showed the following order of substrate preference: methyl sinapate > methyl ferulate > ethyl ferulate. All six FAEs showed poor conversion rates against methyl p-coumarate and methyl caffeate, both of which lacked the methoxy (O-CH3) group substituent on the aromatic ring of the ester substrates, emphasising the requirement for at least one methoxy group on the aromatic ring of the hydroxycinnamic acid ester substrate for optimal FAE activity.
Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Isópteros/microbiologia , Metagenoma , Animais , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Trato Gastrointestinal/microbiologia , Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
The natural aroma compound (+)-nootkatone was obtained in selective conversions of up to 74â¯mol% from inexpensive (+)-valencene substrate by using a comparatively greener biocatalytic process developed based on modifications of the previously published Firmenich method. Buffer identity and concentration, pH, temperature and downstream work-up procedures were optimized to produce a crude product in which >90â¯% of (+)-valencene had been converted, with high chemoselectivity observed for (+)-nootkatone production. Interestingly, the biotransformation was carried out efficiently at temperatures as low as 21⯺C. Surprisingly, the best results were obtained when an acidic pH in the range of 3-6 was applied, as compared to the previously published procedure in which it appeared to be necessary to buffer the pH optimally and fixed throughout at 8.5. Furthermore, there was no need to maintain a pure oxygen atmosphere to achieve good (+)-nootkatone yields. Instead, air bubbled continuously at a low rate through the reaction mixture via a submerged glass capillary was sufficient to enable the desired lipoxygenase-catalyzed oxidation reactions to occur efficiently. No valencene epoxide side-products were detected in the organic product extract by a standard GCMS protocol. Only traces of the anticipated corresponding α- and ß-nootkatol intermediates were routinely observed.
RESUMO
A commercial laccase, Suberase® from Novozymes, was used to catalyse the synthesis of 5,6-dihydroxylated benzo[b]furans and catechol derivatives. The yields were, in some cases, similar to or better than that obtained by other enzymatic, chemical or electrochemical syntheses. The synthesised derivatives were screened against renal (TK10), melanoma (UACC62), breast (MCF7) and cervical (HeLa) cancer cell lines. GI50, TGI and LC50 are reported for the first time. Anticancer screening showed that the cytostatic effects of the 5,6-dihydroxylated benzo[b]furans were most effective against the melanoma (UACC62) cancer cell line with several compounds exhibiting potent growth inhibitory activities (GI50=0.77-9.76 µM), of which two compounds had better activity than the anticancer agent etoposide (GI50 0.89 µM). One compound exhibited potent activity (GI50=9.73 µM) against the renal (TK10) cancer cell line and two exhibited potent activity (GI50=8.79 and 9.30 µM) against the breast (MCF7) cancer cell line. These results encourage further studies of the 5,6-dihydroxylated benzo[b]furans for their potential application in anticancer therapy.
Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Catecóis/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Benzofuranos/síntese química , Benzofuranos/química , Catecóis/síntese química , Catecóis/química , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Células HeLa , Humanos , Lacase/química , Dose Letal Mediana , Células MCF-7 , Neoplasias/patologiaRESUMO
Leaf exudates from Aloe species, such as the Southern African Aloe ferox, are used in traditional medicines for both humans and livestock. This includes aloesin, a skin bleaching product that inhibits the synthesis of melanin. Aloesin, (a C-glycoside-5-methylchromone) can be released from aloeresin A, an ester of aloesin, through hydrolysis. The objective of the current study was to identify an enzymatic hydrolysis method for converting aloeresin A to aloesin, resulting in increased concentrations of aloesin in the aloe bitters extract. More than 70 commercially available hydrolytic enzymes were screened for the conversion of aloeresin A. An esterase (ESL001-02) from Diversa, a lipase (Novozym 388) and a protease (Aspergillus oryzae) preparation were identified during screening as being capable of providing conversion of pure aloeresin A, with the protease giving the best conversion (~100%). It was found that a contaminating enzyme in Novo 388 was responsible for the conversion of aloeresin A to aloesin. This contaminating enzyme, possibly a protease, was able to give almost complete conversion using crude aloe bitters extract, doubling the concentration of aloesin in aloe bitters extract via the hydrolysis of aloeresin A.
Assuntos
Aloe/química , Biocatálise , Cromonas/isolamento & purificação , Cromonas/metabolismo , Glucosídeos/isolamento & purificação , Glucosídeos/metabolismo , Preparações Clareadoras de Pele/isolamento & purificação , África Austral , Aloe/enzimologia , Aspergillus oryzae/enzimologia , Cromonas/química , Esterases/metabolismo , Glucosídeos/química , Hidrólise , Lipase/metabolismo , Peptídeo Hidrolases/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Folhas de Planta/enzimologia , Preparações Clareadoras de Pele/química , Preparações Clareadoras de Pele/metabolismoRESUMO
In the movement to decarbonize our economy and move away from fossil fuels we will need to harness the waste products of our activities, such as waste lignocellulose, methane, and carbon dioxide. Our wastes need to be integrated into a circular economy where used products are recycled into a manufacturing carbon cycle. Key to this will be the recycling of plastics at the resin and monomer levels. Biotechnology is well suited to a future chemical industry that must adapt to widely distributed and diverse biological chemical feedstocks. Our increasing mastery of biotechnology is allowing us to develop enzymes and organisms that can synthesize a widening selection of desirable bulk chemicals, including plastics, at commercially viable productivities. Integration of bioreactors with electrochemical systems will permit new production opportunities with enhanced productivities and the advantage of using a low-carbon electricity from renewable and sustainable sources.
Assuntos
Biotecnologia , Indústria Química , Biocatálise , Combustíveis Fósseis , PlásticosRESUMO
This study describes the design and synthesis of mimetic peptides modelled on the catalytic active site of the fructose-1,6-bisphosphate aldolase (FBPA) enzyme. The synthesized peptides consisting of the turn motifs and catalytic site amino acids of FBPA enzyme were evaluated for catalytic activity in direct asymmetric aldol reactions of ketones and aldehydes. The influence of substrate scope, catalyst loading and solvents including water, on the reaction were also investigated. Nuclear magnetic resonance (NMR) and circular dichroism (CD) were used to determine the secondary structure of the peptides to provide an understanding of the structure-activity relationship. The peptides showed catalytic activity and the aldol products were obtained in low yields (up to 44%), but excellent enantioselectivity (up to 93%) and moderate diastereoselectivity (65 : 35).
RESUMO
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V (max) of 2.03 x 10(-9) s (-1) and a K (m) of 206 microM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70 degrees C and the enzyme had a half life at 60 degrees C of 20.8 h.
Assuntos
Bacillus/enzimologia , Bacillus/genética , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite II Base, from Novozymes. The amine and the p-hydrobenzoquinone was reacted under mild conditions (at room temperature and at 35 degrees C) in a reaction vessel open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoquinone. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/or 5-lipoxygenase inhibiting activity.