Tailoring Microenvironment and Orientation of Immobilized Lactase for Improved Catalysis at Suboptimal pH

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.