Expressão heteróloga de celulases de Myceliophthora heterothallica F.2.1.4 em Pichia pastoris e Escherichia coli com a caracterização e purificação das enzimas produzidas

Os microrganismos termofilicos são de grande interesse biotecnológico, não apenas por tolerar altas temperaturas, mas também por secretarem proteínas de maior estabilidade, com características bioquímicas e estruturais importantes de múltiplas aplicações na indústria. A linhagem do fungo termofílico Myceliophthora heterothallica F.2.1.4, isolada de camas de frango, apresentou alta atividade da enzima β-1,4-endoglucanase (31 U/ml). O gene codificante da β-1,4-endoglucanase de F.2.1.4 foi identificado e expresso em leveduras Pichia pastoris e em bactéria Escherichia coli. A caracterização do gene permitiu identificar esta enzima como membro da família glicosídeo hidrolase 5 (GH5), nomeada neste estudo como Mh_GH5. A enzima apresenta na região N-terminal um módulo de ligação ao substrato (CBM) pertencente a família 1. As enzimas expressas em P. pastoris e em E. coli foram purificadas e submetidas à caracterização bioquímica. A caracterização bioquímica demonstrou que as enzimas recombinantes apresentaram ótimos de temperatura de 55°C (P. pastoris) e 60°C (E. coli) e pH ótimo de 4,0 para ambos os sistemas heterólogos. A análise da estabilidade das enzimas demonstrou que as enzimas expressas por Pichia pastoris apresentaram-se estáveis em temperaturas mais elevadas (70°C), indicando que a glicosilação teria papel essencial na termoestabilidade de enzimas recombinantes. A determinação estrutural em alta resolução foi realizada para o domínio catalítico desta nova β-1,4-endoglucanase GH5 de M. heterothallica expressa em E. coli. A estrutura foi determinada à 1.1 Å e apresenta uma arquitetura frequentemente encontrada dentre os membros da família de GH5, (β/α)8 TIMbarrel. Os dados obtidos neste estudo podem contribuir para a determinação de características envolvidas na termoestabilidade de enzimas provenientes de organismos termofílicos e, consequentemente na obtenção de biocatalisadores eficientes na conversão da biomassa lignocelulósica em biocombustíveis ou compostos químicos biossintetizados.Thermophilic microorganisms have gain attention in the last years because of the biotechnological interest. Essentially these organisms secrete thermostable proteins displaying suitable features, such as biochemical and structural, for industrial platforms. The thermophilic fungus Myceliophthora heterothallica F.2.1.4, isolated from poultry litter, showed high activity for β-1,4- endoglucanase (31 U/ml). A new sequence enconding for glycoside hydrolase was identified and expressed in Pichia pastoris and Escherichia coli. Sequence analysis allowed to classify this new β-1,4-endoglucanase as a member of the glycoside hydrolase 5 (GH5). The enzyme, named Mh_GH5, has N-terminal family 1 carbohydrate binding module (CBM1). The recombinant enzymes expressed in Pichia pastoris and Escherichia coli were purified and subjected to biochemical characterization. Recombinant endoglucanases showed optimal temperature of 55°C (P. pastoris) and 60°C (E. coli) and optimal pH of 4,0 for both heterologous systems. The thermostability of these new enzymes were evaluated and demonstrated that post-translational modifications, such as glycosylation, found on Pichia pastoris heterologous enzymes, contributed to higher thermostability. Structural analysis of the catalytic domain of the new GH5 from M. heterothallica expressed in E. coli was solved at 1.1 Å resolution and shows a common (β/α)8 TIM-barrel fold found in members of GH5 family. Based on the biochemical and structural information we have obtained for Mh_GH5 it is possible to suggest potential features involved in the thermostability of proteins from thermophilic microorganisms and might contribute for the development of new efficient biocatalysts required for biomass conversion to bioethanol and/or chemicals.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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. © 2013 Springer Science+Business Media New York

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 (R). 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 beta-glucosidase