Tailoring Microenvironment and Orientation of Immobilized Lactase for Improved Catalysis at Suboptimal pH
| dc.contributor.author | Fianu, Felicia | en |
| dc.contributor.committeechair | Cheng, Yifan | en |
| dc.contributor.committeemember | Huang, Haibo | en |
| dc.contributor.committeemember | Sun, Wei | en |
| dc.contributor.department | Food Science and Technology | en |
| dc.date.accessioned | 2025-01-16T09:00:31Z | en |
| dc.date.available | 2025-01-16T09:00:31Z | en |
| dc.date.issued | 2025-01-15 | en |
| dc.description.abstract | The U.S. Greek yogurt market has experienced significant growth, rising from 1-2% in 2004 to 40% in 2015, resulting in a large amount of lactose-rich acid whey as a byproduct. Using lactase to transform this waste into valuable products has emerged as a promising solution. Covalent immobilization allows enzymes to be reused and prevents contamination of the product. While immobilizing lactases has been found to enhance their pH and temperature stability, undesired enzyme-substrate interactions can still lead to reduced enzyme activity. This study investigates novel approaches for enhancing the performance of immobilized lactase enzyme through controlled orientation and microenvironment modification. We utilized initiated chemical vapor deposition (iCVD) to fabricate tailored polymeric thin films as enzyme immobilization supports. A site-specific spycatcher/spytag system was employed for direct immobilization of lactase, while polycationic polymers were incorporated to modify the local chemical environment. Fourier Transform Infrared (FTIR) spectroscopy confirmed the retention of key functional groups in the polymeric supports. The epoxide-amine ring-opening reaction between the support and enzyme was verified, indicating covalent immobilization. Directed immobilization resulted in significantly improved enzyme activity compared to random immobilization, particularly at pH 7 and 8. Incorporation of hydrophobic crosslinkers further enhanced the activity of directedly immobilized Lactase, even exceeding that of the free LacZ-ST by 155% at pH 7, while no effect was observed for randomly immobilized LacZ. The inclusion of pH-responsive polycationic moieties in the support enabled LacZ to catalyze at pH 4, where the free enzyme is typically inactive. This study demonstrates the potential of combining controlled enzyme orientation with tailored microenvironments to optimize the performance of immobilized biocatalysts across a broader pH range. | en |
| dc.description.abstractgeneral | With the booming Greek yogurt industry generating substantial amounts of lactose-rich acid whey as a byproduct, there is a pressing need for effective waste management to avoid negative environmental impact. However, the abundance of this acidic byproduct also presents a unique opportunity for product valorization. Lactose, the primary component of acid whey, can be transformed into valuable prebiotics and sweeteners through biotransformation via lactase, an enzyme commonly used for producing lactose-free milk. Nonetheless, the acidic nature of acid whey (~pH 4) inhibits lactase activity, which typically thrives at neutral pH levels (~pH 7). To tackle this challenge, we explored enzyme immobilization that is, fixing the enzymes on a solid support to improve the stability and reusability of lactase under non-ideal pH environment. Our approach involved using initiated chemical vapor deposition (iCVD) to create specialized polymeric supports for enzyme immobilization via covalent bonds. We employed a site-specific immobilization strategy using the spycatcher/spytag system to ensure optimal enzyme orientation, which we hypothesized to be critical for enhancing activity. Additionally, we modified the chemical environment around the immobilized lactase by incorporating a positively charged polymer to allow the local pH to be more neutral than the bulk pH, thus improving the activity of the immobilized lactases. The results showed that our directed immobilization method significantly improved lactase activity, especially at neutral pH levels, compared to immobilized enzymes with random orientation. Furthermore, by adding positively charged components to the immobilization support, we enabled the immobilized lactase to function even at pH 4, where free lactase is completely inactive. This shows promise for using immobilized lactase to process acid whey without pH adjustment. This research highlights the potential for transforming dairy waste into useful products while addressing the limitations of current enzymatic processes, paving the way for more sustainable practices in food production and biomanufacturing. | en |
| dc.description.degree | Master of Science in Life Sciences | en |
| dc.format.medium | ETD | en |
| dc.identifier.other | vt_gsexam:42425 | en |
| dc.identifier.uri | https://hdl.handle.net/10919/124215 | en |
| dc.language.iso | en | en |
| dc.publisher | Virginia Tech | en |
| dc.rights | Creative Commons Attribution 4.0 International | en |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | en |
| dc.subject | Lactase immobilization | en |
| dc.subject | polymers | en |
| dc.subject | biotransformation | en |
| dc.subject | food waste valorization | en |
| dc.title | Tailoring Microenvironment and Orientation of Immobilized Lactase for Improved Catalysis at Suboptimal pH | en |
| dc.type | Thesis | en |
| thesis.degree.discipline | Food Science and Technology | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | masters | en |
| thesis.degree.name | Master of Science in Life Sciences | en |
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