IIT Roorkee uses bacterial enzymes to degrade plasticizers

Plasticizers, which are added to plastics and personal care products, can be absorbed through the skin

Besides plastics, the amount of carcinogenic plasticizers in the environment is increasing at an alarming rate. Plasticizers are chemicals added to plastics and personal care products to enhance flexibility and shine, and are commonly found in items such as baby toys, shampoos, soaps, and food containers. Plasticizers can be absorbed through the skin, making them a direct threat to human health.

A team of researchers headed by Dr. Pravindra Kumar, Professor at the Department of Biosciences and Bioengineering, IIT Roorkee has successfully used an enzyme — esterase enzyme — produced by soil bacteria Sulfobacillus acidophilus to break down diethyl hexyl phthalate (DEHP) plasticizer. While a Chinese team had characterised this enzyme to degrade low molecular weight phthalate diester plasticizers, which can be degraded by several reported esterase enzymes, the IIT Roorkee team has identified its actual potential and used it for degrading difficult to degrade high molecular weight phthalate plasticizers. The research was funded by THDC India Limited, Rishikesh, and the results were published recently in the journal Structure. The group has also discovered that the esterase enzyme can bind to molecules similar to polypropylene used in plastics, making it a potential tool for extracting polypropylene from contaminated water sources.

The esterase enzyme was structurally characterised using X-ray crystallography. “This helped in identifying the active sites of the enzymes and in understanding the detailed mechanism by which this enzyme degrades the DEHP plasticizer,” says Shalja Verma from IIT Roorkee and the first author of the paper. Other sophisticated biochemical and biophysical approaches were also used to understand the efficiency of the enzyme to degrade the plasticizer.

The esterase enzyme remains active for about a month and catalyzes the degradation of DEHP plasticizer with significant efficiency. For large-scale production of this enzyme, the researchers cloned the genes of the EstS1 esterase enzyme into E. coli bacteria and the enzyme was produced in large-scale through aerobic culture.

The enzyme breaks down the DEHP plasticizer into two products — mono-(2-ethylhexyl) phthalate (MEHP) and 2-ethyl hexanol. According to Prof. Kumar, this esterase enzyme, along with other enzymes identified by their group previously can convert high molecular weight phthalate plasticizers into water and carbon-dioxide. And this is where the IIT Roorkee team appears to have an edge.

“The results of our research mark a significant advancement in addressing one of the most pressing environmental challenges — providing a promising path toward a plastic and plasticizer-free future,” says Dr. Kumar. Other researchers involved in the work include Shweta Choudhary, Kamble Amith Kumar, Jai Krishna Mahto, Ishani Mishra, Dr. Ashwani Kumar Sharma, Dr. Shailly Tomar, Dr. Debabrata Sircar and Dr. Jitin Singla.

In 2017, the team isolated another soil bacteria Comamonas testosteroni that breaks down the phthalates produced by DEHP degradation into carbon-dioxide and water. In the lab, the researchers used the enzymes in sequence to first break down DEHP to MEHP and 2-ethyl hexanol using esterase enzyme, which then was degraded to phthalate using another enzyme. The phthalate is then converted to intermediate compounds using a third enzyme (phthalate dioxygenase). The intermediate compound produced after this step is converted into protocatechuate by the enzyme phthalate decarboxylase. Once protocatechuate is produced, the tricarboxylic acid cycle of the bacteria, which is common in all bacteria, converts it to carbon-dioxide and water.

While the esterase enzyme used for breaking down DEHP into MEHP and 2-ethyl hexanol is from Sulfobacillus acidophilus bacteria, the three other enzymes used in sequence are from Comamonas testosteroni bacteria. “In the lab, we have tried using the enzymes in sequence to break down DEHP into water and carbon-dioxide,” says Ms. Verma. “We are now trying to insert the genes of all the five enzymes into bacteria to directly convert the DEHP plasticizer into water and carbon-dioxide.”

Putting all the five enzymes into bacteria will speed up the degradation process not only because the enzymes will act sequentially but also because degradation of the enzymes itself becomes a non-issue once they are integrated into bacteria. The enzymes, whether used for degradation or not, will remain active only for a short time. But once integrated into bacteria, the enzymes remain active for a longer time and the bacteria can be used continuously for degrading the plasticizers. But when the enzymes are used without integrating into bacteria, a fresh batch of enzymes needs to be produced to continue the degradation process. “We are also undertaking enzyme engineering to speed up the degradation process inside the bacteria,” says Ms. Verma.