Aims: Laccase production by the monokaryotic strain Pycnoporus cinnabarinus ss3 was studied using ethanol as inducer in the culture medium.
Methods and Results:The effect of ethanol was tested at 10, 20, 30, 35 and 45 g l )1 and compared with that of ferulic acid, known until now as the most efficient inducer for laccase expression by P. cinnabarinus ss3. In the presence of 35 g l )1 ethanol, laccase activity (266 600 U l )1 ) and productivity (19 000 U l )1 day )1 ) were nine and fivefold higher compared with ferulic acid-induced cultures, and 155-and 65-fold higher compared with noninduced cultures, respectively. In vivo, ethanol added to the culture medium of P. cinnabarinus ss3 favoured a continuous and high expression of laccase gene. Under these conditions, P. cinnabarinus ss3 produced preferentially the isoenzyme LAC I. Ethanol added in vitro to the purified P. cinnabarinus ss3 laccase typically inhibited the enzymatic activity. Conclusions: In spite of an initial inhibitory effect on mycelial growth, ethanol was shown to be a very strong inducer for laccase expression by P. cinnabarinus ss3 allowing an average yield of 1-1AE5 g l )1 laccase.Significance and Impact of the Study: This study identified P. cinnabarinus ss3 as an outstanding producer of laccase in the presence of ethanol as inducer. Ethanol is an inexpensive agricultural by-product and the process is simple to scale-up for industrial production.
In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional~3D! structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature~70 vs. 60 8C! and the apparent T m were increased by about 9 8C, and the mutant was sixfold more stable at 57 8C. The combined mutations A82R0F168H0N169D0D170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R820D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on b-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylañ ϩ3, ϩ2, ϩ1, Ϫ1, Ϫ2!. Y11 and Y16 might represent two additional binding subsites~Ϫ3, Ϫ4! and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.
Two genes encoding family 11 endo-(1,4)-beta-xylanases from Penicillium griseofulvum (PgXynA) and Penicillium funiculosum (PfXynC) were heterologously expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the recombinant enzymes were purified after affinity chromatography and proteolysis. PgXynA and PfXynC were identical to their native counterparts in terms of molecular mass, pI, N-terminal sequence, optimum pH, and enzymatic activity towards arabinoxylan. Further investigation of the rate and pattern of hydrolysis of PgXynA and PfXynC on wheat soluble arabinoxylan showed the predominant production of xylotriose and xylobiose as end products. The initial rate data from the hydrolysis of short xylo-oligosaccharides indicated that the catalytic efficiency increased with increasing chain length (n) of oligomer up to n = 6, suggesting that the specificity region of both Penicillium xylanases spans about six xylose units. In contrast to PfXynC, PgXynA was found insensitive to the wheat xylanase inhibitor protein XIP-I.
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