Seeds of autonomy

Group from Ribeirão Preto creates alternatives for the Brazilian production of enzymes

The moment that Brazilian chemical industries decide to sophisticate today’s modest production of enzymes – a vast class of proteins that are indispensable for the production of foodstuffs, beverages and detergents – they will not have to start from scratch. Nor even to seek knowledge a far. In Ribeirão Preto, a group of biologists is producing at least than 30 enzymes of industrial or scientific interest, many of which are still imported, using about 15 kinds of fungus that act at relatively high temperatures (over 37°C), a rare property that reduces the risk of contamination by other microorganisms. Another innovation: the raw material used is waste from industries or plantations – sugar cane bagasse or corncobs, for example. In comparison with the habitual production methods, which are based on imported sugars, the results are equivalent.

Albeit on a small scale, and subject to adjustments before reaching an industrial scale, the production of the Department of Biology of the Faculty of Philosophy, Sciences and Literature of the University of São Paulo (USP) is even showing some alternatives for the production of noble enzymes like phosphatates, still entirely imported. They are used in testing medicaments and foodstuffs (they differentiate beef or pork) and in molecular biology (they make it possible to piece together the genetic sequences in fragments of DNA). Phosphatases are amongst the most expensive: 100 milligrams of its type, the alkaline ones, costs US$ 300.

At USP in Ribeirão Preto, two teams under the coordination of João Atílio Jorge and Maria de Lourdes Teixeira de Moraes Polizeli have shown that phosphatases can be made – in a much more economical way – using fungi of the Aspergillus genus, which take the shape of ochre grainsafter they grow. There are at least three options: Neurospora crassa, an orange-colored mould, Scytalidium thermophilum and Humicola grisea, in its thermoidea variation, both black. When the fungi feed on cane bagasse, in the proportion of 1 gram of waste for 100 milliliters of culture, they produce enzymes that, as the estimate goes, cost dozens of times less than the imported equivalent.

But it is not just along these lines that Brazilian production, which still has little variety (see table), can be modified. Even the production of routine items can be optimized. This is the case of amylase, widely used in the manufacture of sweets, cookies, medicines and beverages, produced with Aspergillus, Rhizopus, one of the causes of mould in bread, and Neurospora crassa and its mutants, among others.

Bagasse and sugars
In an article published in February in the Journal of Industrial Microbiology e Biotechnology, Maria de Lourdes shows how the method by which Aspergillus phoenicis releases xylanases can be innovated. Xylanes are enzymes that fragment a kind of sugar, xylan, the main component of hemicellulose, which fill the walls of plant cells. According to her, xylanases can reduce the use of chlorine and acids in the production of paper, in just one of their possible industrial uses.

Normally, this enzyme is produced from xylan (US$ 1.50 a gram). When grown in industrial waste, this Aspergillus – separated from the soil and sugar bagasse in the environs of Ribeirão Preto – produced xylosidases at comparable levels: 278.3 units per milligram in xylan and 219.9 in cane bagasse. A further comparison was made with xylose, another imported sugar (US$ 40.00 per 100 grams), with results close to each other: 146.7 units per milligram in xylose and 112.3 in corncobs. There were also successful experiments with rice husks, manioc flour and wheat bran – even with filter paper. Given simple material, but in abundance, the fungi also respond with exuberance.

“We know what the fungi produce and which of them produces what we want”, comments Jorge, summing up the work of at least 20 years of a working party that put together simplicity in its methods and material of work, autonomy, a vision of the whole, and a sound perspective in applying the knowledge born from research. Jorge keeps a collection with about 100 samples of fungi, taken from plants, soil or decaying material. Maria de Lourdes has put together her own collection, now with 30 samples. Not that they are rare: they can be obtained from banks of microorganisms, even without any charge. But, obviously, it is more difficult to discover what they can do.

The two teams work with two categories of fungi: the thermophiles, which develop better in temperatures of over 37° C, like Aspergillus phoenicis, and the mesophiles, whose optimal conditions for growth are found between 20 and 37° C, as is the case of Neurospora. With time, they found out that the thermophilic species of fungi usually produce larger quantities of enzymes than those of the other group. A point in favor of simplicity: the enzymes released by those that favor the heat are also more stable. Ana Carolina Segatto Rizzatti, one of those in the group studying for a doctorate, submitted xylanase to a 60°C bath four-hour long – and there were no modifications.

So resistant, the enzymes from thermophilic fungi can be stored a room temperature and transported without refrigerated chests, which are still indispensable in this area. Another advantage: the level of contamination is low. “The most common bacteria find it difficult to survive in the same temperatures as the thermophilic fungi”, says Maria de Lourdes. The group from Ribeirão Preto also collects findings about the biology of fungi, as a result of the two projects that have FAPESP’s support. Last year, in a study of Chaetomium thermophilum, Jorge proved that the enzyme, maltooligosyl trehalose synthase, is also produced by fungi. It used to be thought that bacteria would be the simplest organisms capable of synthesizing this enzyme, which breaks down trehalose, a kind of sugar that is also present in seaweed, plants and insects.

Sugar of the future
It was already known that trehalose softens the effects of the cold. The novelty is that it can also save plants from excessive heat, according to the results of Ana Carla Medeiros Morato de Aquino thesis for a doctorate with Rhizopus microsporus of the rhizopodiformis variety. Jorge recalls that trehalose is now seen as the sugar of the future: it could be used as a protector of macromolecules – to lengthen the time for which vaccines are valid, for example.

USP’s team is aware not only of the potential, but also of its own limits. “Our competence lies with the laboratory benches”, Jorge guarantees. He knows that it will be necessary to complete a few stages before making industrial applications entirely feasible. One of them is to expand the scale of production while maintaining the yield: every time, the laboratory produces, on average, one liter of enzymes, while the industrial production tanks have, say, one hundred thousand liters. “We have a supermarket, but the customers are missing”, is his comparison.

A supermarket, indeed, that does not stop growing. Last year, Luis Henrique Souza Guimarães, who is studying for his doctorate, went all over the Ilha Solteira region in search of fungi that produce phosphatases. He came back with a smile on his face: one of the finds was a rare Rhizopus microsporus, which grows at over 50°C. It is a respectable producer of phosphatases, when submitted to high temperatures in the molecular biology tests.

Having taught at USP for 23 years, Jorge sees no reason for interrupting his job of identifying and understanding fungi, even if contacts with companies are making headway. He has a good historical perspective. In the 70’s, when the price of oil went up, it became a priority to expand the production of alcohol – and one of the ways taken into consideration was with fungi, he recalls. The price of oil dropped, the interest faded, but research went on. What would he do today, if the problem were to reappear? In reply, the 51 year old researcher takes out of his collection the examples of the extraction of alcohol from cane bagasse, or, from a wider source, cellulose (wood, or even old paper). There may come a moment when the only source of fuel is this”, he says. “The important thing is for us not to lose our mastery of this knowledge.”

From bread and wine to the washing machine
One makes the bread rise, the other makes it crisp. Enzymes are as specific as they are indispensable to the production of foodstuffs – a list that includes cheeses, cookies, jams, beer and wine -, detergents and animal feeds. Brazil still occupies a discrete position, close to 2%, in the world market for enzymes, which is estimated at US$ 1.5 trillion, according to surveys by Institute of Chemistry of the Federal University of Rio de Janeiro (UFRJ) and the Business Communications Company.

Up until now, Brazilian companies have concentrated on the production of the more simple enzymes and those with a wider market, such as amylases and cellulases. Since 1990, the deficit in the balance of trade in enzymes has grown, as a result of the intensive use of reagents for molecular biology and of new applications, such as the manufacture of biodetergents.

UFRJ’s study of this area is being intensified: Brazil is a big producer of raw material for the enzymes that it ends up importing, as it is not able to meet the domestic demand satisfactorily, like bromelain, obtained from pineapple (Ananas sp) , and papain, which comes from the papaw (Carica heterophylla). Both are mainly bought from companies in the United States and Switzerland, to make medicaments.

A similar situation occurs with lysozyme, which is extracted from egg whites, a relatively cheap raw material. It is one of the groups of enzymes with high added value, for medical use, the price of which varies around US$ 62 per kilo. In this case, it is Ireland that meets Brazil’s demand almost in its entirety.

The projects
1. Biochemistry of Fungi: Studies of Enzymes of Filamentous Fungi (nº 95/07446-4); Modality Regular research support line; Coordinator
João Atílio Jorge – USP at Ribeirão Preto; Investment R$ 19,018.30 plus US$ 5,000.00
2. Enzymatic Activities with Biotechnological Potential Produced by Filamentous Fungi of Thermophilic Habits (nº 98/07793-4); Modality
Regular research support line; Coordinator Maria de Lourdes Teixeira de Moraes Polizeli – USP at Ribeirão Preto; Investment R$ 22,099.55 plus U$ 27,026.25