Development of enzymes to cellulose biobleaching: enzymatic personalization service through a robust and innovative molecular engineering process

• Grant number: 10/50328-4
• Support type: Research Grants - Innovative Research in Small Business - PIPE
• Duration: April 01, 2011 - March 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Nínive Aguiar Colonello Frattini
• Grantee: Nínive Aguiar Colonello Frattini
• Company: Verdartis Desenvolvimento Biotecnológico Ltda. - ME
• City: Ribeirão Preto
• Associated scholarship(s): 11/02497-4 - Development of enzymes to cellulose biobleaching: enzymatic personalization service through of a robust and innovative molecular engineering process, BP.PIPE

Abstract

The Brazilian industrial pulp production area is one of the most developed in the world, so that in pulp processing, an enzymatic stage to assist in bleaching pulp must compete with the current, unique processes for each plant of the same company. One of the requirements of the enzymatic stage is the reduction of at least 30% of chlorine dioxide (equivalent to the reduction of 4 kg of the product per ton of cellulose and the reduction of the enzyme selling price, between R$ 10.00 to R$24.00. In order to obtain the highest possible enzymatic efficiency and to attain economic viability, it is necessary to generate enzymes and to produce them from microbial systems that allow maximum production levels to be reached. Currently, the enzymes mode of action for bleaching has been a technical bottleneck for the sector, as the competing enzymes, besides being imported, promote a drastic removal of xylan, with consequent reduction of the quality of the fiber, reduction of yield and increase of the costs of effluent treatment. The company Verdartis has developed a xylanase that has advantages over the competition, as it is an endoxylanase, which hydrolyzes the xylan polymer in the middle of the chain; facilitating the access of oxidants during the process of removal of the Iignin; and releasing less xylan into the effluent. Such xylanase has been developed by the company based on the technique of Directed Evolution (ED). The process involves the introduction of gene diversity by successive cycles of random mutagenesis and construction of libraries containing several variants of the same gene. These variants are analyzed and selected in function of the best performance in relation to a certain enzymatic characteristic related to the variables of the cellulose bleaching process, such as: pH, temperature and pulp structure. After a first selection, the chosen genes are subjected to new cycles of mutagenesis and selection, in order to accumulate mutations favorable to the characteristic of interest. This method allows to create customized enzymes (PersoZyme) to meet the needs of customers and plants with distinct pulp production processes. Achieving the best enzymes in a short time implies broadening the comprehensiveness of the screening and evaluation of gene diversity, generated by ED, from a broad analysis of the phenotypes presented by the bacterial transformants. Manual operations have imposed limits on the evaluation of the diversity generated and access to the best enzymes, requiring a semi-automated process of large-scale ED, aiming at expanding the company's capabilities in the generation, expression and screening of mutant genes of thermotolerant and thermostable xylanases at temperatures between 70-95°C. Thus, a greater number of gene variants will be used in a parallel strategy to improve the xylanases generated by the intrinsic glycosylation of Pichia pastoris, which will be used as a new constitutive system of heterologous expression in search of more efficient and higher production enzymes. (AU)