Heterologous expression of a new lytic polysaccharide monooxygenase from Hahella ganghwensis and their functional characterization.

The powerful class of oxidative enzymes, lytic polysaccharide monooxygenases (LPMOs) - also named Auxiliary Activity (AA) - are able to oxidize recalcitrant polysaccharides on lignocellulosic biomass. In this work, we successfully expressed three catalytic domains from bacterial LPMOs in the yeast Komagataella phaffii: domain MdAA10.1-SD (from Moritella dasanensis), domain VmAA10.2-SD (from Verrucosispora maris), and domain HgAA10.1-SD (from Hahella ganghwensis). Heterologous expression was analyzed by SDS-PAGE, Western-Blot, and Dot-Blot, while functional activity of these proteins was investigated by a combination of mass spectrometric and chromatographic methods. The recombinant LPMO catalytic domain HgAA10.1-SD from H. ganghwensis, a C1-oxidizer, was able to promote an oxidative cleavage of phosphoric-acid swollen cellulose (PASC) substrate in the presence of ascorbic acid as an electron donor, showing its potential for cellulose depolymerization

Produção heteróloga e caracterização funcional de mono­-oxigenases líticas de polissacarídeos de fungos e bactérias.

Mono-oxigenases líticas de polissacarídeos (LPMOs) são enzimas de grande interesse industrial por serem capazes de despolimerizar polissacarídeos recalcitrantes, como a celulose, e apresentarem sinergismo com hidrolases na degradação destes polímeros, tendo potencial para aplicação em diversos processos. Este trabalho teve como objetivo produzir e caracterizar LPMOs recombinantes de origem fúngica e bacteriana e desvendar seu potencial para aplicações biotecnológicas. Três LPMOs dos fungos Neurospora crassa, Gloeophyllum trabeum e Botryobasidium botryosum (subfamília AA9) e cinco LPMOs das bactérias extremófilas Thermobifida fusca, Hahella ganghwensis, Salinispora pacifica, Verrucosispora maris e Moritella dasanensis (subfamília AA10) foram selecionadas e expressas pela levedura metilotrófica Pichia pastoris X­-33 (reclassificada como Komagataella phaffii). As enzimas recombinantes foram caracterizadas combinando ensaio fluorométrico e ensaio de despolimerização de celulose avaliado por cromatografia líquida acoplada a espectrometria de massas. Das oito LPMOs recombinantes obtidas, cinco foram caracterizadas e mostraram -se funcionais por pelo menos um dos métodos utilizados, sendo que três foram capazes de despolimerizar a celulose. A LPMO da bactéria S. pacifica apresentou regioseletividade C1/C4, enquanto as pertencentes a H. ganghwensis e T. fusca apresentaram regioseletividade C1, apresentando potencial para diferentes aplicações biotecnológicas. A capacidade de despolimerizar a celulose faz desse conjunto de LPMOs recombinantes um ativo tecnológico que poderá ser avaliado como insumo para o desenvolvimento de novos produtos/processos que dependem da despolimerização da celulose, p. ex.:, sacarificação de biomassa lignocelulósica para produção de etanol 2G, ração animal, indústria têxtil, entre outras aplicações

Climate, history, society over the last millennium in southeast Africa

Climate variability has been causally linked to the transformation of society in pre-industrial southeast Africa. A growing critique, however, challenges the simplicity of ideas that identify climate as an agent of past societal change; arguing instead that the value of historical climate–society research lies in understanding human vulnerability and resilience, as well as how past societies framed, responded and adapted to climatic phenomena. We work across this divide to present the first critical analysis of climate–society relationships in southeast Africa over the last millennium. To achieve this, we review the now considerable body of scholarship on the role of climate in regional societal transformation, and bring forward new perspectives on climate–society interactions across three areas and periods using the theoretical frameworks of vulnerability and resilience. We find that recent advances in paleoclimatology and archaeology give weight to the suggestion that responses to climate variability played an important part in early state formation in the Limpopo valley (1000–1300), though evidence remains insufficient to clarify similar debates concerning Great Zimbabwe (1300–1450/1520). Written and oral evidence from the Zambezi-Save (1500–1830) and KwaZulu-Natal areas (1760–1828) nevertheless reveals a plurality of past responses to climate variability. These were underpinned by the organization of food systems, the role of climate-related ritual and political power, social networks, and livelihood assets and capabilities, as well as the nature of climate variability itself. To conclude, we identify new lines of research on climate, history and society, and discuss how these can more directly inform contemporary African climate adaptation challenges

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the β-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation

Adaptação de produtos para mercados diferenciados a partir da engenharia reversa

Um dos maiores desafios das empresas exportadoras está na identificação e atendimento, de forma dinâmica, às exigências de mercados externos. Neste artigo, propõe-se uma metodologia para melhoria e adaptação de produtos destinados a mercados diferenciados. A metodologia, implementada em oito passos estruturados, parte de conceitos da Engenharia Reversa e seus habilitadores, bem como de ferramentas para melhoria de processos, tais como QFD - Quality Function Deployment, Cartas de Processo e FMEA - Failure Mode and Effects Analysis. A metodologia proposta é aplicada em um caso prático, onde o objetivo é adaptar um pincel brasileiro às demandas de um distribuidor alemão de ferramentas manuais.<br>Rapid identification and compliance to customized market demands are among the top challenges faced by companies targeting at foreign markets, in this paper we propose an eight-step method for the adaptation and improvement of industrialized products driven by customer demands. The method we propose is grounded on Reverse Engineering principles and process improvement techniques, such as Quality Function Deployment, Process Mapping and Failure Mode and Effects Analysis. The method steps are illustrated by a case example, where the objective is to adapt a paintbrush manufactured by a Brazilian company to the demands of a German distributor of manual tools