The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.

BACKGROUND: Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. RESULTS: In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate. CONCLUSION: This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.

Pectinase activity determination: an early deceleration in the release of reducing sugars throws a spanner in the works!

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

Soil enzyme activities under long-term tillage and crop rotation systems in subtropical agro-ecosystems

Práticas agrícolas que reduzam a degradação do solo e promovam sustentabilidade são importantes para os agrossistemas tropicais/subtropicais. O plantio direto (PD) diminui as perdas de solo e, se combinado com rotação de culturas pode proteger o solo da degradação físico-química provocada pela agricultura intensiva. A atividade enzimática do solo pode fornecer importantes informações de como o manejo do solo está afetando a decomposição da material orgânica e a ciclagem dos nutrientes. Assim, avaliou-se a atividade das enzimas amilase, celulose, arilsulfatase, fosfatase ácida e fosfatase alcalina em um experimento a campo, instalado em 1976 em Londrina, PR, que tem como tratamentos o preparo do solo (plantio direto ou convencional) nas parcelas e a rotação de culturas (soja/trigo, milho/trigo e algodão/trigo) nas subparcelas. Amostras de solos foram coletadas a 0-5, 5-10 e 10-20 cm de profundidade em 1997 e 1998. Na profundidade de 0-5 cm sob PD, observaram-se aumentos de 68 per center na atividade da amilase, 90 per center na celulase, 219 per center na arilsulfatase, 46 per center na fosfatase ácida e 61 per center na fosfatase alcalina. Observaram-se correlações significativas entre a atividade enzimática e o C-orgânico total do solo e o C e N da biomassa microbiana. Esses resultados evidenciam que a atividade enzimática do solo é um indicador sensível de alterações na qualidade do solo, promovidas pelo manejo.