Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l⁻¹ in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest.     

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

Transgenic rice as a novel production system for <Emphasis Type="Italic">Melanocarpus</Emphasis> and <Emphasis Type="Italic">Pycnoporus</Emphasis> laccases

Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1-1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].     

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Fractionation of subcellular membranes of the secretory pathway from the peroxidase-producing white-rot fungus Phanerochaete chrysosporium

Abstract Mycelia from the basidiomycete Phanerochaete chrysosporium , producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.     

Shifting the biotransformation pathways of L-phenylalanine into benzaldehyde by Trametes suaveolens CBS 334.85 using HP20 resin

AIMS: The biotransformation of L-phenylalanine into benzaldehyde (bitter almond aroma) was studied in the strain Trametes suaveolens CBS 334.85. METHODS AND RESULTS: Cultures of this fungus were carried out in the absence or in the presence of HP20 resin, a highly selective adsorbent for aromatic compounds. For the identification of the main catabolic pathways of L-phenylalanine, a control medium (without L-phenylalanine) was supplemented with each of the aromatic compounds, previously detected in the culture broth, as precursors. Trametes suaveolens CBS 334.85 was shown to biosynthesize benzyl and p-hydroxybenzyl derivatives, particularly benzaldehyde, and large amounts of 3-phenyl-1-propanol, benzyl and p-hydroxybenzyl alcohols as the products of both cinnamate and phenylpyruvate pathways. CONCLUSION: The addition of HP20 resin, made it possible to direct the catabolism of L- phenylalanine to benzaldehyde, the desired target compound, and to trap it before its transformation into benzyl alcohol. In these conditions, benzaldehyde production was increased 21-fold, from 33 to 710 mg l-1 corresponding to a molar yield of 31%. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the good potential of Trametes suaveolens as a biotechnological agent to synthesize natural benzaldehyde which is one of the most important aromatic aldehydes used in the flavour industry.