Geetanjali S. Wakade
Shina Lin
Uma Kumari
Henry Daniell
| Geetanjali S. Wakade | Shina Lin | Uma Kumari | Henry Daniell |
Geetanjali S. Wakade, Shina Lin, Uma Kumari, Henry Daniell
Basic and Translational Sciences, University of Pennsylvania, School of Dental Medicine
Introduction
Microfibers (diameter > 5mm) released from textiles are ubiquitous, cause pollution and are harmful to aquatic flora and fauna,animals, and human life. Each laundry cycle releases~9 million microfibers into wastewater treatment plants that cannot be filtered. Household electric dryers contributes 40% more release of microfibers than that of washing machines into the atmosphere.More than 90% of anthropogenically modified cellulose microfibers were found in the sediments of deep arctic sea and major lakes in North America. Denim microfibers contribute 60% of the total anthropogenically modified cellulose microfibers observed in these sediments. Chemical dyes are carcinogenic and toxic to the ecosystem. Therefore, biological degradation of microfibers and indigo dye is investigated.
Methods
Codon Optimized sequences of Cp-EG1, Cp-bgl1, Cp-Swo1, Cp-Cutinase and Cp-Lac were inserted into marker—free chloroplast vectors and stably integrated into chloroplast genomes via bombardment.Freeze dried plant biomass was used as a source of each enzyme. Enzyme cocktails were designed through dose response curve to obtain maximum sugar release. Microfiber hydrolysis was carried out at 50°C for 96 hrs.Indigo decolorization was conducted at room temperature for 1 hour using laccases through dose response curve for enzyme and indigo dye.
Results
• Enzymes required for biodegradation of microfibers and indigo dye are expressed in transplastomic tobacco plant by chloroplast engineering technology.Stable integration of genes coding for endoglucanases, beta-glucosidases, swollenin, cutinase and laccase into chloroplast genomes and homoplasmy were confirmed by Southern blots. Enzyme assays for endoglucanase, beta-glucosidase and laccase revealed activities 1092, 454.6 and 10454 µmol/hr/g of protein respectively. Microfiber hydrolysis using 1% commercial enzymes + 8% plant enzymes showed 73% of sugar release efficiency and 70% of reduction in use of commercial enzyme load. Decolorization (up to 90%) was obtained using 0.25% plant enzyme powder of 50 ppm Indigo dye.
Conclusion
Enzymes expressed in plants do not need expensive fermentation and purification.Use of plant enzymes significantly reduces cost on commercial enzymes. Released sugar can serve as a platform for synthesis of commercially significant biochemicals and biofuels. Enzymatic detoxification of dye using plant enzymes lowers the severity of toxicity for disposal into the water bodies.