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Good bacteria

Enzymes degrade proteins and PET bottles and may have new industrial uses

eduardo cesarHippopotamus and other zoo animals may be the source of useful bacteriaeduardo cesar

Although useful and present in our day-to-day lives, PET packaging, which is used for water and soft drinks, creates a serious environmental problem. Every year more than 50% of the national production, estimated at some 380,000 tons, is deposited in  landfills where it remains for years on end until its total decomposition. The rest is used in a variety of recycling processes, resulting in other products such ropes, carpets and handicraft items. But good news from Japan indicates that very soon a new form of microbiological degradation from this type of packaging may take place. Studies along these lines are being carried out by researcher Kohei Oda, emeritus Professor at the Kyoto Institute of Technology, and one of the forerunners, in the seventies, of the discovery of a protease inhibitor, known as pepstatine, a type of protein whose function is to break down other proteins in order to activate or deactivate them; many years later this same protein was used to inhibit a proteolytic enzyme (formed by proteases) of HIV. He succeeded in producing a consortium of bacteria that degraded PET packaging, a polymer made from a poly resin (ethylene terephthalate) in just eight weeks and in half this time using a specific bacteria isolated from this consortium. These microorganisms, which are not pathogenic, secrete a variety of enzymes that decompose the polymer. The characteristics of the bacteria and the enzymes responsible for the rapid degradation are being kept secret because of a contract with the Kyoto Institute of Technology.

“Microbial degradation offers the possibility of renewing places where the packaging is buried or has accumulated over the years and is an alternative capable of competing financially with chemical degradation”, says Oda. He was in São Paulo from April to October this year, as a visiting professor at the Federal University of São Paulo (Unifesp). He also lectured and held meetings at other São Paulo research institutes. The bacteria he identified in landfills in Japan metabolize polyesters, such as PET. Polyesters are polymers whose chemical bonds result from the bond of an acid with  alcohol. These bonds, called esters, can be undone by treating them with acid, or even with enzymes. “If degradation occurs by means of a group of bacteria or by a single bacteria, enzymes are produced, which are the proteases or proteolytics that hydrolyze (break the bond in the presence of water) the ester bonds and after degrade the monomers, which are the units that comprise the plastics, forming carbon dioxide and water”, explains Luiz Juliano, a Professor at the Department of Biophysics at Unifesp.

The biggest challenge to making the process that was discovered by the Japanese microbiologist commercially viable (talks are under way with some companies in Japan) is to solve the issue of how to break up the crystallinity of PET, without which the bacteria cannot decompose the packaging. When poly (ethylene terephthalate) is processed to form the bottles it acquires a crystalline consistency that prevents any interaction between the bottle walls and water. This property is beneficial, because it allows PET packaging to be used for preserving and packaging drinks, but at the same time makes it difficult to degrade. According to Oda, the answer to breaking up the crystalline structure of PET lies with heating it to 260° Celsius (C) in autoclaves or treating it with microwaves. The heat breaks down the material’s crystallinity and leaves ester bonds more exposed to hydrolysis, making the decomposition process possible.

Maria de Lourdes Teixeira Polizeli/USPFormations of Rhisopus microsporus fungus, a producer of amylase, an enzyme used in the production of sweetenersMaria de Lourdes Teixeira Polizeli/USP

Multiple applications
The use of microorganisms for degrading products that pollute the environment (a process known as bioremediation) is just one of the many areas of applied microbiology. This branch of science aims at prospecting and using microorganisms that produce enzymes, or other substances, in the broadest range of activities, such as the medicine and food, tanning, cosmetics, fabrics, sugar and alcohol, and paper and pulp industries. Today, one of the focuses of microbiology is the fermentation of sugars for the production of  biofuels. “Brazil has also made contributions to the microbiology of human pathology, such as the Chagas disease, leprosy, tuberculosis and dengue fever, but because of its biodiversity it offers many possibilities for the advance of microbiology in other areas”, says Juliano. “Brazil is waking up to its potential”, says the Brazilian researcher and coordinator of a project in this area, whose aim is to identify enzymes from humans, fungi and bacteria, as well as animal poisons with industrial or pharmacological use. He points to the work of Professor Maria de Lourdes Teixeira de Moraes Polizeli, from the Biology Department of the Ribeirão Preto School of Philosophy, Science and Arts of the University of São Paulo (USP). She has isolated a variety of fungus, Rhisopus microsporus, from mushroom compost that has proven to be an excellent producer of amylasis. This enzyme is used in the production of sweeteners, glucose syrup for brewers and the drugs industry. The most interesting thing is that these fungi grow in very cheap solid substrates, such as corncobs and cane bagasse, and at temperatures over 45°C, making industrial production easy.

One of the projects of Juliano’s group concerns celiac disease, i.e., human intolerance to gluten. This problem arises from the fact that gluten has a protein called gliadin, which is very rich in proline, one of the twenty natural amino acids that make up proteins. This makes gliadin very hard to digest, making hydrolysis by the digestive enzymes of the stomach and intestines difficult. Undigested fragments of this protein can be absorbed and many people (one out of every two hundred, in Western countries) build up clinical intolerance. “We’re synthesizing hundreds of peptidic subtracts (protein fragments) containing proline to examine the proteolytic enzymes that have a preference for proline. This will allow us to look for highly efficient proteases when it comes to degrading gliadin fragments”, says the researcher from Unifesp. The final objective of the project, however, is to discover enzymes that can be used in the treatment of gluten or administered in the form of capsules to patients who suffer from gluten-intolerance.

Another important project that the group from Unifesp is engaged in is focusing on a proteolytic enzyme isolated from the bacteria that grow in fermented fish products in Thailand. Professor Oda and Thai scientists are collaborating in this research. What is the importance of this enzyme? “Sardines are fermented in extremely high concentrations of cooking salt, the substrate in which the bacteria live”, replies Juliano. “The proteases secreted by these bacteria also operate in highly saline concentrations, of more than 20%. Industry wants enzymes of this type, as they can stand salinity and high temperatures”, he emphasizes. Brazil has plants that grow alongside salt pans and researcher João Lúcio de Azevedo, from the Luiz de Queiroz College of Agriculture (Esalq) at the University of São Paulo (USP), has indicated the possibility of the endophytic bacteria (that live in plant tissue) from these plants expressing proteases that operate in high salt concentrations. In order to find microorganisms like those that operate in the fermentation of sardines or that have other medical, biological or industrial applications, researchers are involved with a real piece of detective work. They go into the field and look through different environments in search of these microscopic beings.

Animal riches
Prospecting for microorganisms was one of the reasons for the visit of Luiz Juliano and Kohei Oda to the São Paulo Zoological Park Foundation last October. A little while ago Oda isolated nearly 500 strains of bacteria, whose common property is producing lactic acid or acetic acid, from the feces of animals in zoos in Japan. Generally, these bacteria are not pathogenic or toxic and they can produce enzymes or other substance that are biologically interesting.

Juliano believes that equally rich, diverse and abundant material may be found in the zoo in São Paulo, where animal feces are mixed with wood-chips and other plant remains and composted. “In the composting process the temperature reaches 70°C. We can find fungi and bacteria growing under extreme temperature conditions”, he says. “I believe we have an opportunity to look for an enormous variety of microorganisms coming from different animal species.” And this all happens without the need to touch or disturb the animals.

Besides the zoo, Juliano and Oda visited other microbiology research institutions in the city of São Paulo and in the state to propose setting up a cooperative network in the area, such as with USP in Ribeirão Preto, with Paulista State University in São José do Rio Preto and Rio Claro, with the State University of Campinas, and with the Unifesp campus in Diadema. The association of the research groups of Professor Juliano and Professor Oda will expand the studies into the proteases already discovered, while also providing an opportunity for disclosing to other interested groups the experiments in prospecting for microorganisms that are of practical use. “We also want to make a contribution to the area of proteolytic enzymes, by making the proteases study platform that our group has put together over the last 27 years available to others”, says Juliano.

The Projects
1.Peptidic substrates and inhibitors for proteolytic enzymes
Thematic project
Luiz Juliano Neto – Unifesp
R$ 376,320.26 and US$ 111,757.59 (FAPESP)

2. Kohei Oda, Kyoto Institute of Technology, Japan
Helping with visiting researcher travel costs
Luiz Juliano Neto – Unifesp
R$ 41,193 (FAPESP)