Littérature scientifique sur le sujet « Structural analysis and biochemical characterization of lipases »

In order to determine the potential of biochemical and structural features of Elaeis guineensis Jacq. oil palm mesocarp lipases, the LIP2 gene was isolated, expressed, purified and characterized through the Escherichia coli microbial recombinant system. Gene analysis of LIP2 revealed that it is composed of 1584 base pairs which are encoded in 528 amino acid residues with a molecular weight of around 57 kDa. LIP2 has distinctive lipolytic properties in terms of α/β fold and the catalytic triad for lipase. The LIP2 lipase was successfully expressed and purified from E. coli Rosetta (DE3) via affinity chromatography. The optimal temperature and pH for the lipase activity was 30 °C and a pH of 9, respectively. Stability was profoundly increased with the addition of metal ions (Ca2+, Mg2+, Mn+, and Ni+), along with organic solvents (ethanol and octanol). pNP myristate was the most suitable among all pNP esters. In biophysical characterization analysis, LIP2 has a thermal denaturing point at 66 °C, which mostly consists of random patterns (39.8%) followed by α-helix (30.3%), turns (23.8%) and β-sheet (6.2%). From the successful purification and characterization, the potential of oil palm mesocarp lipase was able to be further explored.

Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

ABSTRACT A direct involvement of the PreS domain of the hepatitis B virus (HBV) large envelope protein, and in particular amino acid residues 21 to 47, in virus attachment to hepatocytes has been suggested by many previous studies. Several PreS-interacting proteins have been identified. However, they share few common sequence motifs, and a bona fide cellular receptor for HBV remains elusive. In this study, we aimed to identify PreS-interacting motifs and to search for novel HBV-interacting proteins and the long-sought receptor. PreS fusion proteins were used as baits to screen a phage display library of random peptides. A group of PreS-binding peptides were obtained. These peptides could bind to amino acids 21 to 47 of PreS1 and shared a linear motif (W1T2X3W4W5) sufficient for binding specifically to PreS and viral particles. Several human proteins with such a motif were identified through BLAST search. Analysis of their biochemical and structural properties suggested that lipoprotein lipase (LPL), a key enzyme in lipoprotein metabolism, might interact with PreS and HBV particles. The interaction of HBV with LPL was demonstrated by in vitro binding, virus capture, and cell attachment assays. These findings suggest that LPL may play a role in the initiation of HBV infection. Identification of peptides and protein ligands corresponding to LPL that bind to the HBV envelope will offer new therapeutic strategies against HBV infection.

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/β hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

Abstract Previous studies have determined that various Qa2 serologic determinants can be removed from the surface of spleen cells by treatment with a phospholipase C. Our studies have determined that the class I molecule Qa2, expressed on the surface of spleen cells and activated T cells, behaves as an integral membrane protein based on its ability to associate with detergent micelles. Studies utilizing two purified phospholipase C have revealed that although most (90 to 95%) of the Qa2 molecules expressed on the surface of resting spleen cells are released as intact 40-kDa polypeptides associated with beta 2-microglobulin, activated T cells contain a major cell subpopulation expressing lipase-resistant Qa2 molecules. Flow cytometric analysis revealed that L3T4+-activated T cells expressed lipase-sensitive Qa2 molecules, whereas Lyt-2+ cells express lipase-resistant forms of the Qa2 molecule. The relationship between the secreted form of the Qa2 molecule and the lipase-generated soluble Qa2 molecule was investigated. Based on SDS-PAGE analysis, the secreted Qa2 molecules has a Mr of 39 kDa whereas the cell surface form released from either resting spleen or activated T cells by phosphatidylinositol-specific phospholipase C has a Mr of approximately equal to 40 kDa. Furthermore, the secreted Qa2 molecule lacks an epitope, cross-reacting determinant, often present on lipase-solubilized cell surface molecules. Thus, based on serologic and biochemical criteria, the soluble Qa2 molecules generated by an exogenous phospholipase C and the secreted Qa2 molecule are structurally distinct.

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

A causa del rapido esaurimento dei combustibili fossili, dell’aumento dei prezzi e dei problemi ambientali connessi al loro utilizzo, la ricerca di combustibili alternativi sta attirando un crescente interesse. Il biodiesel, una miscela di monoalchil esteri di oli vegetali o grassi animali, è un potenziale sostituto del gasolio ottenuto da petrolio. Esso, infatti, presenta diversi vantaggi rispetto al convenzionale combustibile diesel: bassa tossicità, alta biodegradabilità, minore emissione di particolato atmosferico, ottenimento da fonti di energia rinnovabili. La reazione di produzione del biodiesel è una transesterificazione di trigliceridi di origine vegetale o animale con alcoli a catena corta (metanolo o etanolo) e il processo comunemente impiegato è la catalisi chimica in condizioni alcaline. Questo processo, però, prevede un alto consumo energetico e produce una notevole quantità di acque di scarico alcaline che devono essere opportunamente trattate. Tali problematiche hanno spostato l’interesse verso la biocatalisi, che prevedere l’utilizzo di enzimi come catalizzatori di reazioni altamente selettive e condotte in condizioni blande e poco inquinanti. Le lipasi sono gli enzimi più adatti a catalizzare la transesterificazione di oli vegetali in esteri monoalchilici di acidi grassi a lunga catena. La stechiometria della reazione prevede l’utilizzo di metanolo (MeOH) in rapporto molare 3:1 rispetto all’olio. Il MeOH, però, è spesso causa dell’inattivazione del biocatalizzatore per motivi che non sono ancora compresi a fondo. In questo lavoro sono stati studiati gli effetti prodotti dal metanolo su lipasi già impiegate o potenzialmente utilizzabili/di interesse per la produzione industriale di biodiesel. Preliminarmente, è stato messo a punto e validato un nuovo metodo analitico di Spettroscopia a Infrarossi in Trasformata di Fourier (FTIR) per rilevare e quantificare la presenza di metil esteri ed acidi grassi in miscele di reazione complesse. Questo metodo, più semplice e rapido della più comune tecnica di gas cromatografia (GC), è stato applicato per monitorare reazioni enzimatiche di transesterificazione e idrolisi in cui metil esteri ed acidi grassi sono prodotti/substrati di reazione. Il metodo si basa sulla rilevazione del picco a 1435 cm-1 in derivata seconda dello spettro FTIR, la cui intensità è direttamente proporzionale alla quantità di metil esteri. Al contempo è possibile monitorare l’idrolisi dei trigliceridi misurando l’assorbimento a 1709 cm -1, lunghezza d’onda al quale assorbe l’acido oleico (Paper I). L’indagine sugli effetti di inattivazione causati dal metanolo è stata realizzata su due diverse lipasi, da Burkholderia glumae (BGL) e Candida antartica (CALB), in due diversi sistemi di reazione. Innanzitutto, è stata studiata l’influenza del metanolo sulla attività catalitica e sulla conformazione di BGL, enzima noto per essere tollerante al metanolo. Sono state allestite miscele di reazione a diversi rapporti molari trioleina:MeOH (da 1:1 a 1:6), in cui MeOH è presente in quantità sia maggiore che inferiore rispetto alla stechiometria 1:3 richiesta dalla reazione. Il massimo di attività catalitica è stato rilevato in presenza di ~70% di metanolo, corrispondente ad un rapporto molare trioleina:MeOH 1:5, mentre per rapporti molari superiori la resa cala drasticamente. L’idrolisi, invece, è risultata proporzionale al contenuto di acqua presente nella miscela di reazione. Parallelamente, è stata condotta una indagine sull’impatto del metanolo su struttura e conformazione dell’enzima. In particolare, è stata analizzata la struttura secondaria e terziaria di BGL in presenza di concentrazioni variabili di alcool utilizzando le tecniche di spettrometria di massa, spettroscopia di dicroismo circolare e fluorescenza intrinseca. Nel saggio di transesterificazione, nelle condizioni in cui BGL esprime il massimo di attività catalitica, ovvero in presenza del 50-70% di alcool in fase acquosa, il MeOH stesso influenza la stabilità dell’enzima provocandone una graduale denaturazione e successiva aggregazione (Paper II). Infine, è stato razionalizzato l’effetto del metanolo e il ruolo dell’attività dell’acqua sulla attività catalitica di CALB. Questo enzima è uno dei più impiegati nella produzione industriale di biodiesel, principalmente nella forma immobilizzata (Novozyme 435). E’ stato proposto un meccanismo molecolare dell’inattivazione da metanolo applicando i dati sperimentali ad un modello cinetico ed effettuando simulazioni di dinamica molecolare. Le condizioni sperimentali più adeguate a quelle simulate con metodi computazionali corrispondono ad una reazione di metanolisi di vinilacetato (VA) che produce metilacetato e vinil alcool. Le reazioni sono state realizzate a VA costante, con un range di concentrazione di MeOH da 0.7% a 60% v/v, a tre diversi valori di attività dell’acqua (aw): 0.02, 0.05 e 0.09. Per ciascuna aw saggiata, la velocità iniziale di CALB è massima alla più bassa concentrazione di MeOH impiegata, diminuisce drasticamente all’aumentare del MeOH, e si mantiene costante a concentrazioni di alcool superiori al 10%. E’ emerso, invece, che l’attività dell’acqua non ha effetto sulla diminuzione di attività catalitica indotta da MeOH. Questi risultati sperimentali sono stati adattati ad un modello cinetico e combinati alle simulazioni di CALB in miscele ternarie toluene/MeOH/VA. Ne è stato ottenuto un modello termodinamico del comportamento di CALB da cui emerge che il MeOH agisce sull’enzima come un inibitore competitivo (Paper III).
The search for alternative fuels has gained much attention because of the rapid depletion of fossil fuels, their rising prices and environmental concerns. Biodiesel, monoalkyl esters of vegetable oils or animal fats, is a potential substitute for petroleum-based diesel. It exhibits several advantages over diesel fuel such as low toxicity, high biodegradability, lower emission of particulate matter and its derivation from renewable energy sources. The process commonly used to produce commercial biodiesel is the chemical alkaline catalysis to convert vegetable oils or fats and methanol (MeOH) to fatty acid methyl esters (FAME). However, this process is energy consuming and produces high amount of alkaline waste water. This motivates the current interest toward biocatalysis. Among enzymes, lipases convert vegetable oils to FAME in highly selective reactions carried out in mild, environmentally-favorable conditions. Different lipases have been described suitable for biodiesel production. The enzymatic approaches have become more and more attractive but their industrial exploitation is impaired by relatively high costs, partly due to the short operational life of catalysts. A major cause of low lipases performance is the inhibition by methanol. Although many scientific publications proved that the activity of several free and immobilized lipases is severely affected by methanol, none of them addresses the causes of inactivation. These investigations focused on how to override the inhibition rather than to explain why inhibition occurs. In the present work we investigated the molecular and kinetic effects of methanol on lipases already used or potentially applicable/interesting for the industrial production of biodiesel. Firstly, we set up a novel method based on analytical Fourier Transform Infrared Spectroscopy (FTIR) to detect and quantify the total methyl esters and fatty acids present in complex mixtures. The FTIR approach allows to monitor simultaneously transesterification and hydrolysis reactions catalyzed by lipase enzymes and exhibits several advantages over the traditional analytical methods: rapid, inexpensive, accurate, requiring very limited sample preparation and simple statistical analysis of the spectroscopic data. This method was validated through the comparison with data obtained by gas chromatography, a conventional technique commonly employed for the determination of methyl esters. Our FTIR method is based on the determination of the intensity of two different peaks, proportional to the total methyl ester and oleic acid amounts present in the mixture (Paper I). The study of the inactivation effects exerted by methanol was carried out on two different lipases, the Burkholderia glumae lipase (BGL) and the Candida antartica lipase B (CALB), in two different reaction systems. First of all we investigated the influence of methanol on the catalytic activity and conformation of BGL, a lipase known in literature to be tolerant to methanol. To this aim, 24-hours transesterification reactions of triolein and methanol, at 37°C and different oil:MeOH molar ratios, ranging from 1:1 up to 1:6, were carried out. MeOH is thus present from lower to higher amounts than required by the reaction stoichiometry (1:3). We found that the highest catalytic activity is reached in the presence of ~70% v/v MeOH, corresponding to a molar ratio 1:5, while for higher molar ratios the yield decreases dramatically. On the other hand, hydrolysis is proportional to the water content. In parallel, a structural study of the impact of MeOH on BGL structure and conformation was performed by means of mass spectrometry (MS), circular dichroism (CD), and intrinsic fluorescence spectroscopy. Under the conditions providing the highest reaction yield, we found that the enzyme stability is perturbed, leading to gradual protein unfolding and finally to aggregation (Paper II). Morevorer, we investigated the effect of methanol and water activity on CALB, one of the most commonly employed lipases for the industrial production of biodiesel, mainly in its immobilized form (Novozym 435). We obtained a model of the molecular mechanism of CALB deactivation exerted by methanol. Experimental data from the enzymatic alcoholysis reaction of vinyl acetate (VA) by methanol in the presence of toluene were fitted to a kinetic model. Reactions at constant VA (15.2% v/v), methanol concentrations ranging from 0.7% up to 60% v/v and at three different water activity values (0.02, 0.05, 0.09) were performed. CALB shows the highest activity at MeOH concentrations as low as 0.7% followed by a sharp decrease at higher concentrations. For MeOH concentrations higher than 10% the activity was constant. Water activity does not influence the decrease of lipase activity induced by MeOH. Experimental results were adapted to a kinetic model and combined to molecular dynamics simulations of CALB in toluene–methanol–water mixtures. Thus, a thermodynamic model of the lipase activity was established, which indicates that methanol acts as a competitive inhibitor of the enzyme (Paper III).

As endo-1,4-?-xilanases (EC 3.2.1.8) formam o maior grupo de enzimas hidrolíticas envolvido na degradação da xilana, visto que catalisam a hidrólise aleatória de ligações glicosídicas do tipo ?-1,4 no interior da sua cadeia principal, produzindo xilooligossacarídeos de diferentes tamanhos. Na natureza, essas enzimas estão intimamente relacionadas ao fornecimento de energia para o desenvolvimento dos organismos que as produzem. Em geral, as xilanases são isoladas preferencialmente de bactérias e fungos, e têm demonstrado grande potencial na produção de pães, ração animal, alimentos, bebidas, xilitol e bioetanol. O presente trabalho propôs o isolamento de uma nova endo-1,4-?-xilanase por meio de técnicas de produção e purificação acessíveis que pudessem viabilizar economicamente a integração desse biocatalisador aos processos industriais. O fungo Aspergillus tamarii Kita, oriundo de uma amostra de solo da Mata Atlântica, mostrou-se um bom produtor de xilanases em meio de cultura Adams suplementado com bagaço de cevada, um subproduto das indústrias cervejeiras. Após a otimização do processo de fermentação submersa, o extrato enzimático exibiu duas xilanases em gel de atividade para proteínas nativas, identificadas por espectrometria de massas como glicosil hidrolases pertencentes às famílias 10 e 11. A sacarificação enzimática de três resíduos agroindustriais, com base em um delineamento experimental de misturas, demonstrou que a combinação ternária desses componentes, em iguais proporções, possui considerável relevância para a produção de açúcares fermentáveis, tais como glicose e xilose. Em ensaios de imobilização, a xilanase GH11 foi satisfatoriamente estabilizada em matrizes de caráter iônico e covalente. A imobilização por ligação covalente multipontual em glioxil-agarose elevou a temperatura ótima de atividade de 60 para 65 °C e ofertou um considerável ganho de termoestabilidade ao derivado, que apresentou meia vida de 60 minutos a 80 °C. Além disso, a estabilização da enzima nesse suporte permitiu a produção dos seguintes xilooligossacarídeos: xilobiose, xilotriose, xilotetraose e xilopentaose. A purificação da xilanase GH11 foi realizada por meio de uma única etapa cromatográfica de troca catiônica, com rendimento final de 36,72% e um fator de purificação de 7,43 vezes. A massa molecular da enzima foi estimada em 19,5 kDa. Ademais, a sua estrutura tridimensional foi predita por modelagem comparativa, exibindo como modelo final uma arquitetura do tipo ?-jelly roll, comum às xilanases da família 11. Em ensaios de caracterização, a xilanase apresentou melhor atividade em pH 5,5 e manteve atividade residual superior a 80% na faixa de pH compreendida entre 4,0 e 9,0, durante 24 horas. Em relação à temperatura, a sua atividade ótima foi observada a 60 °C, contudo, a sua termoestabilidade foi mais expressiva a 50 °C, retendo cerca de 70% da sua atividade inicial por 480 minutos. Para a xilana beechwood, os valores de velocidade máxima e constante de dissociação aparente foram iguais a 1.330,20 µmol/min/mg e 8,13 mg/mL, respectivamente. Na concentração de 5 mM, os metais pesados Co2+, Hg+, Pb2+ e Zn2+ apresentaram um ponderável efeito de inibição sobre a xilanase GH11, enquanto que os íons Ba2+ e Ni2+, assim como os compostos ?-mercaptoetanol e DTT, exibiram um aumento superior a 20% em sua atividade. Por fim, a análise em tempo real da atividade xilanásica revelou que o substrato xilopentaose corresponde ao menor xilooligossacarídeo capaz de ser eficientemente hidrolisado. Sendo assim, a nova endo-xilanase GH11 isolada do fungo A. tamarii Kita exibe uma série de propriedades físico-químicas favoráveis a sua aplicabilidade em escala industrial.
The endo-1,4-?-xylanases (EC 3.2.1.8) form the largest group of hydrolytic enzymes involved in the degradation of xylan, since they catalyze the random hydrolysis of ?-1,4 glycosidic bonds within the main chain of this polysaccharide, producing xylooligosaccharides of different sizes. In nature, these enzymes are closely related to supplying energy for the development of the organisms that produce them. In general, xylanases are preferentially isolated from bacteria and fungi, which show great potential in industries as brewing, animal feed, food, beverage, xylitol and bioethanol. The present work proposed the isolation of a new endo-1,4-?-xylanase by available techniques of production and purification that can economically make feasible the integration of this biocatalyst to industrial processes. The fungus Aspergillus tamarii Kita, obtained from a soil sample of the Atlantic Forest, showed to be a good xylanase producer in Adams culture medium supplemented with barley bagasse, a byproduct of breweries. After the optimization of the submerged fermentation process, the crude enzymatic extract exhibited two xylanases in activity gel for native proteins, identified by mass spectrometry as glycosyl hydrolases belonging to families 10 and 11. The enzymatic saccharification of three agroindustrial residues, based on an experimental mixture design, showed that the ternary combination of these components, in equal proportions, has considerable relevance for the production of fermentable sugars, such as glucose and xylose. The xylanase GH11 was satisfactorily stabilized on matrices of ionic and covalent character in immobilization assays. Covalent multipoint immobilization on glyoxyl agarose raised its optimum temperature of activity from 60 to 65 °C and offered a considerable gain in thermostability to the derivative, which presented a half-life of 60 minutes at 80 °C. In addition, enzyme stabilization on this support allowed the production of the following xylooligosaccharides: xylobiose, xylotriose, xylotetraose and xylopentaose. Xylanase GH11 purification was carried out by means of a single cation exchange chromatographic step, with final yield of 36.72% and purification factor of 7.43 times. The molecular mass of this xylanase was estimated as 19.5 kDa. Moreover, its three-dimensional structure was predicted by comparative modeling, exhibiting a ?-jelly roll type folding as a final model, common to xylanases of family 11. In characterization tests, xylanase presented better activity at pH 5.5 and was considerably stable in the pH range of 4.0 to 9.0. Regarding temperature, its optimum activity was observed at 60 °C, however, its thermostability was more expressive at 50 °C, retaining about 70% of its initial activity for 480 minutes. In the presence of beechwood xylan, the values of maximum velocity and the constant of apparent dissociation were 1,330.20 µmol/min/mg and 8.13 mg/mL, respectively. At concentrations of 5 mM, the heavy metals Co2+, Hg+, Pb2+ and Zn2+showed an inhibition effect on the xylanase, whereas Ba2+ and Ni2+ ions, as well as ?-mercaptoethanol and DTT, exhibited an increase of more than 20% in their activity. Finally, the real-time analysis of xylanase activity revealed that the xylopentose substrate corresponds to the lowest xylooligosaccharide capable of being hydrolyzed. Thus, the new endo-xylanase GH11 isolated from the fungus A. tamarii Kita exhibits a series of physicochemical properties favorable to its applicability on an industrial scale.

博士
國立成功大學
生命科學系
107
Fungal laccase is a blue, glycosylated four-copper oxidase that catalyzes the oxidation of phenolic units in lignin as well as a number of phenolic compounds and aromatic amines. A high-efficiency laccase, DLac, was secreted by an indigenous fungal strain Cerrena sp. RSD1 isolated from rice straw compost, it is identified using 18S rDNA and internal transcribed spacer sequencing analysis. A procedure of submerged culture using rice straw as a feedstock was carried out to produce DLac with high catalytic efficiency of 1.5×109 s-1 M-1 show the enzyme to be diffusion-limited. So far only a few study focus on structural properties of the diffusion-limited enzyme, it is important to investigate the crucial structural features that govern the enzyme kinetics. The crystal structure of DLac was determined at 1.38 Å resolution. DLac displays the typical fungal laccase architecture consisting of three domains, and each domain is folded into the greek key β-barrel topology. Four copper atoms were coordinated with His and Cys residues to create the catalytic site. The crystal structure was determined to atomic resolution and its overall structure was found to be closely homologous to the monomeric laccases. However, DLac displays some unique substrate-binding loops different from those in other laccases. The substrate-binding residues with small side-chains and the short substrate-binding loop IV widen the substrate-binding cavity and this may facilitate access by larger substrates. DLac is not as highly-glycosylated as other fungal laccases and contains one highly-conserved glycosylation site at N432 and another unique site at N468. The N-glycans stabilize the substrate-binding loops and the protein structure and the first N-acetylglucosamine is crucial for catalytic efficiency. A submerged culture method useful for industrial application allows a five-fold increase of protein yield to be achieved. Laccases that are tolerant to organic solvents are powerful bio-catalysts with broad applications in biotechnology, most frequently carried out at high concentration of solvent, during which process the proteins can be unfolded and enzyme activity can be lost. In this study it will be shown that pre-incubation of fungal laccases with organic solvents can result in an effective (and reversible) 1.5 to 4.0 fold enhancement of enzyme activity. Several organic solvents, including acetone, methanol, ethanol, DMSO, and DMF were effective in this specific enhancement in all the laccases studied. The enhancement was not substrate-specific and could be observed in both phenolic and non-phenolic substrates. Although laccase pre-incubated with organic solvents was sensitive to high temperature, it remained stable at 25°C, which was an advantage for long term storage. The 3-D structure of DLac, pre-incubated with acetone, was determined and it was confirmed that the protein structure remained intact and stable at high concentrations of organic solvent. Furthermore, the turnover rate of fungal laccases was improved by pre-incubation in an organic-solvent and DLac showed the highest enhancement among the fungal laccases examined. This investigation has shed light on improving fungal laccase usage under extreme conditions and has extended the opportunities for bioremediation, decolorization, and organic synthesis.