Leo RamosA new type of cheaper and more efficient, environment-friendly enzyme will soon be available on the market. The enzyme was developed by the Verdartis Desenvolvimento Biotecnológico company, which stemmed from a research group at the University of São Paulo (USP) in Ribeirão Preto. The advantages of the new product include the need for less water to process the wood for pulp, and less chlorine dioxide, the chemical agent employed to bleach the wood. In addition, the enzyme can be tailor-made according to the characteristics and needs of each paper manufacturer. To develop the enzyme, Verdartis counted on the funds of five projects funded by FAPESP’s Innovative Research at Small Companies Program (Pipe).
Marcos Roberto Lourenzoni, one of the partners, has a Chemistry Degree from the University of São Paulo’s (USP) School of Philosophy, Sciences and Literature of Ribeirão Preto (FFCLRP). He explains that pulp is comprised of fibrous elements that are individualized in the industrial processing of wood and acquire a brownish color due to the existence of ligno compounds. Bleaching is a major step to make these fibers appropriate for the manufacturing of paper. High-cost chemical compounds that require special care when they are discarded as industrial waste are used for bleaching. In this process, two chemical substances – lignin and uronic acids – are left over. These two substances are responsible for the consumption of the reagents in the fibers. Their content can be reduced by using enzymes.
Chlorine dioxide reacts with the lignin – which causes the brownish coloring – breaking it into smaller, soluble molecules that are extracted in the process. A significant amount of this lignin remains encapsulated in the pulp. This requires higher quantities of chlorine dioxide and other reagents to access it. “The enzymes are specific and act to break down the substrate and other components of pulp, such as the residual lignin,” Lourenzoni explains. “This creates pores in the pulp, allowing the chlorine dioxide to have easier access to the lignin. As a result, the process needs smaller amounts of chemical products to achieve the same effect, and therefore costs are reduced.”
In addition to reducing bleaching costs, the enzymes developed by Verdartis can also reduce electric power costs. Electric power is used to refine the pulp. At this stage, pulp is diluted in water and goes through a pulp refiner to remove the fibers from the macrofibrils; this mechanical process is run on electric power. The action of the enzymes breaks up clumps of pulp, facilitating the refinement and consequently reducing electric power consumption.
Enzymes are proteins produced by live organisms that act as catalysts; that is, they accelerate chemical reactions. However, in order to be used in any industrial process, enzymes have to be produced in large quantities, with the specific and desired characteristics, within a short period of time. Various pulp-bleaching enzymes are available on the market. The problem is that they are very expensive and release a large quantity of organic material into the wastewater. This requires treatment, which in turn increases costs.
The technology developed by Verdartis provides a solution to deal with this situation. By using molecular (or genetic) engineering, the company produces personalized catalysts, based on such microorganisms as the Escherichia coli. These catalysts are tailor-made to each of the clients’ industrial process or factory. “In other words, we create a process-specific enzyme instead of modifying the process for the enzyme,” says Lourenzoni. “In addition, our enzyme does not release waste into the water, which means it is more environmentally and economically efficient.”
The techniques used to create this enzyme include the so-called controlled evolution, or evolutionary biotechnology. Controlled evolution is faster than Nature. It reproduces the evolution of natural biodiversity in the lab, by means of the same environment-adapting selection mechanism. In practice, controlled evolution imitates events that happen in nature, that is, in natural evolution (mutation, recombination and natural selection). The difference is that the enzyme’s desired properties are pre-defined by the scientists.
In this technique, random mutations are induced in the DNA, resulting in a “ library” of modified genes, in which each gene creates a code for the catalyst. “This library contains millions of possibilities or combinations, among which are some good solutions to express the desired characteristic for a given enzyme,” Lourenzoni explains. “In practice, a library of the genes of wild enzymes is created. Copies of these genes are inserted into expression vectors, used to transform the microorganism. The E. coli is the vector in the case of the enzyme created by Verdartis.”
Genes for evolution
Thus, a group of bacteria, each one with a specific gene, will produce a different enzyme. A group of such genes is selected, representing a tiny quantity in proportion to all the combinations found in the library, because it is impossible to process and test all the possibilities that are generated. Thus, it is possible to come up with enzymes adapted to a given environment that simulate the desired industrial conditions, and select the enzymes that will function in such an environment. The next step is to sequence these enzymes’ DNA and check the mutations. “The genes of the better enzymes can be submitted to recombination, aiming at evolution,” Lourenzoni explains.
However, this is not enough. Although controlled evolution is much faster than natural evolution, it is unable to sustain the personalized enzyme business model. The process to create a given enzyme can take months, but the market needs new enzymes immediately. This is why the pace of the process has to speed up. To this end, Verdartis developed software called Artizima, used to deal with the astronomical number of enzymes evaluated experimentally. This software makes it possible to speed up controlled evolution cycles, minimizing the development time of a specific catalyst for a given function.The last step in the development of this technology is laboratory-scale production and subsequent industrial phase that the company has not achieved yet. Lourenzoni explains that this process is divided into two steps: the first step is production and the second step, referred to as downstream, entails the separation and purification of the enzyme.
Bases of transformation
This biotechnology was initially developed during the time that Robert Ruller, advised by professor Richard John Ward, of the Chemistry Department of FFCLRP, was working on his doctorate thesis. Lourenzoni himself went to work at a technological company after he got his doctorate degree. When working at this company, Lourenzoni was contacted by Ward, who wanted to know if Lourenzoni would be interested in the technology. Lourenzoni was not interested, as at the time she was focusing on information technology. “ So in 2006 we decided to open another company to transform Ruller and Ward’s research work into technology,” says Lourenzoni.
At the time, Verdartis participated in business plan competitions organized by the Supera Incubator for Technological Companies, created in 2003, thanks to a partnership between USP, the Institute for Advanced Studies on Health Foundation (Fipase), the City Government of Ribeirão Preto and Sebrae. “We were the winners in 2007; and one of the awards was the right to an office at the incubator facility, whose premises were very small and space was eagerly sought after,” recalls Verdartis’ director. “We opened the company in June that year, after we had submitted a project, which I coordinated, to the Pipe program.”
Funds provided by FAPESP helped the company develop technologies based on the technology transferred from Ward’s laboratories. Other projects funded by the Pipe program followed. The second project, approved in 2009, focused on the development of a bioprocess to produce enzymes. Still in 2009, Verdartis got approval from FAPESP to develop a third project under the Pipe program. The third project entailed the development of software to develop in silico (in a computer) catalysts. The company also obtained funds from the Funding Agency for Studies and Projects (Finep) and from its partners.
In 2011, the company started working on a fourth project, also under the Pipe program. The project is still being worked on. The objective is to improve the catalytic efficiency of enzymes at high temperatures and pHs ranging from 8 to 10. In 2012, Verdartis qualified for a fifth project under the Pipe-III tender. The objective of this Project is to develop part of the scale of the enzyme production bioprocess. The project is being sponsored by Suzano Papel e Celulose S/A, which is interested in the outcome. The next step is to build a facility with the capacity to produce any kind of catalyst, by fermenting any microorganism. To this end, Verdartis is talking to potential investment partners.
Suzano’s evaluation is that the enzymes developed by the small company from Ribeirão Preto have a future. “We have already tested most of the enzymes available on the market and we have always encountered problems,” says, Augusto Fernandes Milanez, the company’s production, research, and development consultant. “The two main problems are the high price and the great quantity of organic material they release into the wastewater.” This is why Suzano is supporting and guiding Verdartis in the development of the catalysts. In addition, Suzano is supplying Verdartis with the necessary pulp to conduct tests and result analyses. “The people in this company have made promising developments,” says Milanez. “We see evolution every time we see the product. This is why we see potential in the enzymes made by Verdartis.”
These catalysts have a potential market as well. Brazil is one of the world’s biggest and most advanced paper manufacturers. The sector is comprised of 222 operational paper mills and 2.2 million hectares of forests planted for industrial purposes. According to data from the Brazilian Pulp and Paper Association (Bracelpa), in 2011, the industry’s export revenues came to US$ 7.2 billion, a 6.2% increase in comparison to 2010. Of this amount, US$ 5 billion corresponds to pulp exports, which accounted for 69.5% of the industry’s total exports last year. As these figures show, there will be plenty of opportunities on the market for bleaching enzymes.
1. Persozyme – personalized enzymes created for controlled evolution in the biobleaching of wood pulp (nº 2007/51561-1); Modality Pipe – Innovative Research in Small Companies; Coordinator Marcos Roberto Lourenzoni – Verdartis; Investment R$ 261,866.47 and US$ 72,891.00 (FAPESP)
2. Artizima – computer tool for the in silico evolution of enzymes used in biorefining (nº 2007/59308-3); Modality Pipe – Innovative Research in Small Companies; Coordinator Renato Luis Tame Parreira – Verdartis; Investment R$ 79,120.80 (FAPESP)
3. Enzyme production bioprocess for biomass biorefining: pulp bleaching (nº 2008/53426-7); Modality Pipe – Innovative Research in Small Companies; Coordinator Alvaro de Baptista Neto – Verdartis; Investment R$ 141,712.46 and US$ 22,582.85 (FAPESP)
4. Enzyme development for pulp bleaching: enzyme personalization service by means of a robust and innovative molecular engineering process (nº 2010/50328-4); Modality Pipe – Innovative Research in Small Companies; Coordinator Ninive Aguiar Frattini – Verdartis; Investment R$ 262,280.68 and US$ 145,863.76 (FAPESP)
5. Enzyme production bioprocess for biomass biorefining: pulp bleaching (nº 2011/51096-2); Modality Pipe – Innovative Research in Small Companies; Coordinator Alvaro de Baptista Neto – Verdartis; Investment R$ 64,463.72 and US$ 38,212.41 (FAPESP)