ABSTRACT
AIMS: The aim of the study was to characterize 10 hemicellulolytic enzymes obtained from a wheat straw-degrading microbial consortium. METHODS AND RESULTS: Based on previous metagenomics analyses, 10 glycosyl hydrolases were selected, codon-optimized, synthetized, cloned and expressed in Escherichia coli. Nine of the overexpressed recombinant proteins accumulated in cellular inclusion bodies, whereas one, a 37·5-kDa protein encoded by gene xylM1989, was found in the soluble fractions. The resulting protein, denoted XylM1989, showed ß-xylosidase and α-arabinosidase activities. It fell in the GH43 family and resembled a Sphingobacterium sp. protein. The XylM1989 showed optimum activity at 20°C and pH 8·0. Interestingly, it kept approximately 80% of its ß-xylosidase activity in the presence of 0·5% (w/v) furfural and 0·1% (w/v) 5-hydroxymethylfurfural. Additionally, the presence of Ca2+ , Mg2+ and Mn2+ ions increased the enzymatic activity and conferred complete tolerance to 500 mmol l-1 of xylose. Protein XylM1989 is also able to release sugars from complex polysaccharides. CONCLUSION: We report the characterization of a novel bifunctional hemicellulolytic enzyme obtained through a targeted synthetic metagenomics approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The properties of XylM1989 turn this protein into a promising enzyme that could be useful for the efficient saccharification of plant biomass.
ABSTRACT
Hy-Line W-36, W-98, and Brown hens lay approximately the same number of eggs/hen housed to 80 wk; however, little is known about differences in performance during heat stress (HS). Two experiments were performed. The first experiment evaluated intestinal calcium uptake (CaT), heat shock protein-70 (HSP70) liver expression, and endocrine status in the 3 strains under heat stress in response to 1 h of transient exposure to high temperature before onset of 18 h of HS. The second experiment evaluated the differences between W-36 and W-98 in acid-base status observed at 2 different ambient temperatures. The HSP70 and CaT data were analyzed as a completely randomized design (CRD) using a 3 x 2 factorial with strain as a 1 factor and preexposed and control treatments as the other. Estrogen and progesterone data were analyzed as a CRD using repeated measures in a 3 x 2 x 2 factorial with strain as a the first factor, preexposure and control treatments as the second factor, and phase of blood collection as the third factor. The data of the second experiment were analyzed as a CRD using repeated measures in a 2 x 2 x 2 factorial with strain, temperature, and phase of blood collection as the factors. The method applied in both experiments was based on the mixed model (SAS). The results show a strain effect, with the higher CaT in the W-36. The results indicated that transient exposure to HS did not induce changes in HSP70 liver expression. In the second experiment, the blood gas values did not differ between strains, except for the partial pressure of CO(2), in which the values at 22 degrees C are higher for the W-36. At 38 degrees C, there was an increase in blood pH and a reduction in HCO(3)(-) in both strains. The results indicate that endocrine, acid-base status, and Ca homeostasis represent important factors to be considered in assessing genetic differences for thermotolerance.
