Characterization, expression and application of a zearalenone degrading enzyme from Neurospora crassa.

A gene named zenc, encoding a zearalenone lactonase from Neurospora crassa, was over-expressed in Pichia pastoris. The zenc gene is 888-bp in length, encoding a 295-residue polypeptide. Purified ZENC has maximal activity at pH 8.

Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5.

Background: Plant biomass, the most abundant natural material on earth, represents a vast source of food and energy in nature. As the main component of plant biomass, xylan is a complex polysaccharide comprising a linear β(1,4)-linked backbone of xylosyl residues substituted by acetyl, arabinosyl, glucuronysyl and 4-O-methylglucuronycyl residues.

Results: Aspergillus fumigatus Z5 is an efficient plant biomass depolymerization fungus.

Cloning and characterization of the first GH10 and GH11 xylanases from Rhizopus oryzae.

The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086.

Genome mining for new α-amylase and glucoamylase encoding sequences and high level expression of a glucoamylase from Talaromyces stipitatus for potential raw starch hydrolysis.

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P.

Alternaria alternata as a new fungal enzyme system for the release of phenolic acids from wheat and triticale brans.

This study describes the release of antioxidant ferulic acid from wheat and triticale brans by mixtures of extracellular enzymes produced in culture by a strain FC007 of Alternaria alternata, a dark mold originally isolated from Canadian wood log. The genus of the mold was confirmed as Alternaria by 18S ribosomal DNA characterization. Enzyme activities for feruloyl esterase (FAE) and polysaccharide hydrolyzing enzymes were measured, and conditions for release of ferulic acid and reducing sugars from the mentioned brans were evaluated.

Cold adaptation of a mesophilic cellulase, EG III from Trichoderma reesei, by directed evolution.

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase III (EG III) from Trichoderma reesei, and obtained a cold adapted mutant, designated as w-3.

A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae.

A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R.

Mining Xanthomonas and Streptomyces genomes for new pectinase-encoding sequences and their heterologous expression in Escherichia coli.

Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv.

Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature-guided sequence alignment.

In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (Tm) of the enzyme more than 1 to 2 degrees C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PL(Xc)) produced a 6 degrees C increase in Tm and a 23-fold increase in the half-life at 45 degrees C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established Tm values.

Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor.

A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain.

Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products.

Pulps with residual lignin ranging from 6.4-27.4% (w/w) were prepared from mixed softwoods using a proprietary biorefining technology (the Lignol process) based on aqueous ethanol organosolv extraction.

Microplate-based filter paper assay to measure total cellulase activity.

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers.

Effects of sugar inhibition on cellulases and beta-glucosidase during enzymatic hydrolysis of softwood substrates.

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and beta-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both beta-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on beta-Glucosidase activity.