Heterologous expression, purification and biochemical characterization of a new xylanase from Myceliophthora heterothallica F.2.1.4.

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-ß-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching.

Evaluation of microwave-assisted pretreatment of lignocellulosic biomass immersed in alkaline glycerol for fermentable sugars production.

A pretreatment with microwave irradiation was applied to enhance enzyme hydrolysis of corn straw and rice husk immersed in water, aqueous glycerol or alkaline glycerol. Native and pretreated solids underwent enzyme hydrolysis using the extract obtained from the fermentation of Myceliophthora heterothallica, comparing its efficiency with that of the commercial cellulose cocktail Celluclast®. The highest saccharification yields, for both corn straw and rice husk, were attained when biomass was pretreated in alkaline glycerol, method that has not been previously reported in literature. Moreover, FTIR, TG and SEM analysis revealed a more significant modification in the structure of corn straw subjected to this pretreatment. Highest global yields were attained with the crude enzyme extract, which might be the result of its content in a great variety of hydrolytic enzymes, as revealed zymogram analysis. Moreover, its hydrolysis efficiency can be improved by its supplementation with commercial ß-glucosidase.

Yeast diversity isolated from grape musts during spontaneous fermentation from a Brazilian winery.

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR-RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.