Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli

AbstractA gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg−1. By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6–7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg−1) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM−1s−1). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h

Primjena oksidoreduktaza iz gljiva roda Trametes spp. u biotehnologiji – obilje katalitičkog djelovanja

Those oxidoreductases that are part of the ligninolytic complex of basidiomycete and ascomycete fungi have played an increasingly important role in biotechnological applications during the last decade. The stability of these extracellular enzymes, their good solubility, and a multitude of catalyzed reactions contribute to this trend. This review focuses on a single genus of white-rot basidiomycetes, Trametes, to highlight the numerous possibilities for the application of this microorganism as well as three of its enzymes: laccase, cellobiose dehydrogenase, and pyranose 2-oxidase. Whereas laccase is without doubt a major player in biotechnology, the two other enzymes are less well known, but represent emerging biocatalysts with potential. Both cellobiose dehydrogenase and pyranose 2-oxidase are presumed to participate in lignin breakdown and will be used to exemplify the potential of less prominent oxidoreductases from this genus.Posljednjih deset godina oksidoreduktaze koje sudjeluju u procesu razgradnje lignina gljiva stapčara i mješinarki imaju sve važniju ulogu u biotehnološkoj primjeni. Tomu trendu je pridonijela stabilnost tih ekstracelularnih enzima, njihova dobra topljivost i mogućnost primjene u raznim katalitičkim procesima. U ovom je revijalnom prikazu osobito opisana jedna vrsta gljive stapčare koja uzrokuje bijelu trulež, Trametes, i istaknute razne mogućnosti primjene toga mikroorganizma i njegovih triju enzima: lakaze, celobioza-dehidrogenaze i piranoza-2-oksidaze. Dok lakaza bez sumnje ima glavnu ulogu u biotehnologiji, druga su dva enzima manje poznata, ali sve se više koriste kao biokatalizatori. Pretpostavlja se da celobioza-dehidrogenaza i piranoza-2-oksidaza sudjeluju u razgradnji lignina te se pomoću njih mogu objasniti mogućnosti manje poznatih oksidoreduktaza toga roda gljiva

Primjena oksidoreduktaza iz gljiva roda Trametes spp. u biotehnologiji – obilje katalitičkog djelovanja

Those oxidoreductases that are part of the ligninolytic complex of basidiomycete and ascomycete fungi have played an increasingly important role in biotechnological applications during the last decade. The stability of these extracellular enzymes, their good solubility, and a multitude of catalyzed reactions contribute to this trend. This review focuses on a single genus of white-rot basidiomycetes, Trametes, to highlight the numerous possibilities for the application of this microorganism as well as three of its enzymes: laccase, cellobiose dehydrogenase, and pyranose 2-oxidase. Whereas laccase is without doubt a major player in biotechnology, the two other enzymes are less well known, but represent emerging biocatalysts with potential. Both cellobiose dehydrogenase and pyranose 2-oxidase are presumed to participate in lignin breakdown and will be used to exemplify the potential of less prominent oxidoreductases from this genus.Posljednjih deset godina oksidoreduktaze koje sudjeluju u procesu razgradnje lignina gljiva stapčara i mješinarki imaju sve važniju ulogu u biotehnološkoj primjeni. Tomu trendu je pridonijela stabilnost tih ekstracelularnih enzima, njihova dobra topljivost i mogućnost primjene u raznim katalitičkim procesima. U ovom je revijalnom prikazu osobito opisana jedna vrsta gljive stapčare koja uzrokuje bijelu trulež, Trametes, i istaknute razne mogućnosti primjene toga mikroorganizma i njegovih triju enzima: lakaze, celobioza-dehidrogenaze i piranoza-2-oksidaze. Dok lakaza bez sumnje ima glavnu ulogu u biotehnologiji, druga su dva enzima manje poznata, ali sve se više koriste kao biokatalizatori. Pretpostavlja se da celobioza-dehidrogenaza i piranoza-2-oksidaza sudjeluju u razgradnji lignina te se pomoću njih mogu objasniti mogućnosti manje poznatih oksidoreduktaza toga roda gljiva

Structural Comparison of Different Galacto-oligosaccharide Mixtures Formed by beta-Galactosidases from Lactic Acid Bacteria and Bifidobacteria

The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhβ-gal) and Lactobacillus plantarum (Lp609Lhβ-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus, together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)- and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ∼90% of all GOS components. GOS mixtures obtained with β-galactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS)

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Nicht verfügbarMultigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucosemethanolcholine (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.(VLID)192910

Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete <it>Glomerella cingulata </it>is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by <it>Glomerella </it>(<it>anamorph Colletotrichum) spp</it>.</p> <p>Results</p> <p>Heterologous expression of active <it>G. cingulata </it>GDH has been achieved in both <it>Escherichia coli </it>and <it>Pichia pastoris</it>, however, the expressed volumetric activity was about 4800-fold higher in <it>P. pastoris</it>. Expression in <it>E. coli </it>resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a <it>P. pastoris </it>transformant resulted in an expression of 48,000 U L^-1of GDH activity (57 mg L^-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in <it>P. pastoris </it>and the wild-type enzyme from its natural fungal source.</p> <p>Conclusions</p> <p>The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in <it>P. pastoris </it>facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies.</p

Quantitative transcript analysis of the inducible expression system pSIP: comparison of the overexpression of Lactobacillus spp. β-galactosidases in Lactobacillus plantarum

<p>Abstract</p> <p>Background</p> <p>Two sets of overlapping genes, <it>lacLMReu </it>and <it>lacLMAci</it>, encoding heterodimeric β-galactosidases from <it>Lactobacillus reuteri </it>and <it>Lactobacillus acidophilus</it>, respectively, have previously been cloned and expressed using the pSIP vector system and <it>Lactobacillus plantarum </it>WCSF1 as host. Despite the high similarity between these <it>lacLM </it>genes and the use of identical cloning and expression strategies, strains harboring <it>lacLMReu </it>produced about twenty-fold more β-galactosidase than strains containing <it>lacLMAci</it>.</p> <p>Results</p> <p>In this study, the plasmid copy numbers (PCN) of expression vectors pEH9R (<it>lacLMReu</it>) and pEH9A (<it>lacLMAci</it>) as well as the transcription levels of both <it>lacLM </it>genes were compared using quantitative PCR methods. Analyses of parallel fermentations of <it>L. plantarum </it>harboring either pEH9R or pEH9A showed that the expression plasmids were present in similar copy numbers. However, transcript levels of <it>lacLM </it>from <it>L. reuteri </it>(pEH9R) were up to 18 times higher than those of <it>lacLM </it>from <it>L. acidophilus </it>(pEH9A). As a control, it was shown that the expression levels of regulatory genes involved in pheromone-induced promoter activation were similar in both strains.</p> <p>Conclusion</p> <p>The use of identical expression strategies for highly similar genes led to very different mRNA levels. The data indicate that this difference is primarily caused by translational effects that are likely to affect both mRNA synthesis rates and mRNA stability. These translational effects thus seem to be a dominant determinant for the success of gene expression efforts in lactobacilli.</p

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

<p>Abstract</p> <p>Background</p> <p>Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-<smcaps>D</smcaps>-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, <it>i.e</it>. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).</p> <p>Results</p> <p>A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-<smcaps>D</smcaps>-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed β-mannanase, from <it>Aspergillus niger </it>BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL) in <it>Pichia pastoris</it>. The enzyme was secreted by <it>P. pastoris </it>and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent K_mvalues of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-β-<smcaps>D</smcaps>-mannan (from carob) are 0.6 mg mL^-1, 2.0 mg mL^-1, 2.2 mg mL^-1and 1.5 mg mL^-1, respectively, while the k_catvalues for these substrates are 215 s^-1, 330 s^-1, 292 s^-1and 148 s^-1, respectively. Judged from the specificity constants k_cat/K_m, glucomannan is the preferred substrate of the <it>A. niger</it> β -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.</p> <p>Conclusion</p> <p>This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-β-mannosidase from <it>A. niger </it>in <it>Pichia pastoris</it>. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant β-mannanase will be valuable in various biotechnological applications.</p

Heterologous expression of a recombinant lactobacillal -galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system

Background: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric -galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri -galactosidase. Results: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum -galactosidase levels obtained were 130 U/mg protein and 3540 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), -galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant -galactosidase. Conclusions: The over-expression of recombinant L. reuteri -galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.(VLID)90704