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To effectively immobilize an enzyme while maintaining its high activity and stability, the design of supports with controlled geometric structures and heterogeneous surface properties is desirable. Towards this goal, heterogeneous titanium dioxide (TiO2) surfaces with controlled pore sizes were synthesized in this study and used to efficiently immobilize lipase. The immobilized lipase activity increased by a factor of 1.31 with an increase in the TiO2 pore size from 11.46 to 21.14 nm. The highest protein loading of 15.52 mg/g was achieved in ethenyl triethoxy silane (ETS)-modified TiO2 (E-P25) after immobilizing the enzymes at 30 ℃, pH 7.33 for 3 h and using a protein concentration in solution of 0.176 mg/mL. Among the different surface functionalities, the highest activity yield of 428.04 % was accomplished by using TiO2 calcinated at 650 ℃ and modified by ETS as immobilization support. The immobilized enzymes showed excellent storage stability and retained almost 95 % of their activity after being stored at 4 ℃ for 8 weeks. This research provides experimental evidence that highlights the importance of studying of enzyme immobilization on the supports with synergistically designed geometric structures and surface chemical properties.

Bacterial cellulose (BC) is a biodegradable material with many excellent properties for enzyme immobilization. However, hydroxyl groups of low reactivity in glucose units cannot directly react with the amines of enzymatic proteins. In this work, amino-functionalized bacterial cellulose was firstly used as carrier to immobilize horseradish peroxidase (HRP) via glutaraldehyde coupling. SEM, FT-IR, XPS, BET, and TGA were used to characterize the properties of modified BC and showed that it is more suitable for enzyme immobilization. The optimum pH range for immobilized HRP (pH 5.5–8.5) was wider than that of free enzyme (pH 6–8), and the immobilized HRP exhibited good adaptability to environmental alkalinity. The relative activity of immobilized HRP at 25–40 °C was greater than 90%, significantly surpassing that of free HRP. Furthermore, the obtained kinetic constant values showed that modified BC had decreased affinity for the substrate. Additionally, modified BC-immobilized HRP was reused efficiently for 10 cycles with greater than 70% of its original activity retained. Under optimal conditions, coupling ratio and specific activity could reach 86.7% and 41.7 Ug -1 min -1 , respectively. These results show that the immobilization of HRP on amino-functionalized BC enhanced its appropriateness for a future use in various biotechnological and environmental applications.

Surface amine modification could not only improve the microenvironment near the active sites of enzyme, but also enhance the multi-point chemical crosslinking between the enzyme and carrier. The linear structured polymer of e-poly- l -lysine (EPL) is an ideal donor of amino with much more exposed on the surface for enzyme attachment. Analysis of the result of dynamic light scattering (DLS) and circular dichroism (CD) demonstrated the favorable electrostatic interactions and negligible impact on the conformation of enzymes, Glucose Oxidase and Catalase (GOx&CAT). Titration and dissociation curve together with Zeta-potential characterization revealed that enzymes (EPL@GOx&CAT) under the protection of EPL had more stable structure and better activity and stability in acidic reaction environment. Meanwhile, the activity recovery of immobilized EPL@GOx&CAT increased to 1.56 times higher than that of GOx&CAT, and the acid resistance was improved by 1.44 times with the optimum pH shifting to acidic by 0.50 unit. The substrate affinity was raised with the decrease of Km from 5.98 to 4.35 mg mL−1. Thus, the gluconic acid production in low pH system could be increased by pH-engineering of the enzyme microenvironment via conjugation with positively charged polyelectrolyte.

Orientation immobilization of enzymes has attracted intensive interest owing to the retainable specific activity and stability. Specially, glycoprotein immobilized onto Concanavalin A (Con A) modified carriers induces the orientation of the enzyme. However, the effects of the interface properties of carriers and enzymes are still not well understood yet. In this study, we synthesized the activated porous poly (styrene- divinylbenzene) resin carriers with 30 nm pore sizes and 72 m2 g−1 specific surface areas and decorated with Con A. The resultant loading capacity of NP1 on Con A modified carriers was as high as 4.02 mg g−1 wet support as a result of strong affinity between the enzyme and Con A decorated on carriers. It was found that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on Con A modified porous carriers were significantly improved. The reduction of Km from 18.40 ± 0.55 mg mL−1 to 17.19 ± 0.51 mg mL−1 illustrated the improved substrate affinity of HA-GA-ConA-NP1. Moreover, Con A-affinity NP1 exhibited the best operational stability that only 7% of its initial activity was lost even after 9 batches repeated reaction. This work demonstrates that surface property manipulation of porous carriers and its derivatives has great potential in efficient biocatalytic systems.

Microbial nuclease P1 from Penicllium citrinum was immobilized on macroporous absorbent resins: strong polar poly (styrene-co-DVB) resin (SPPSD), polymethacrylic ester resin and poly (styrene-co-DVB)-Br resin. The results showed that SPPSD was the best carrier. Three methods of glutaraldehyde cross-linking were used and simultaneous immobilization and cross-linking (CIS) was demonstrated to be the best method. The functional properties of immobilized nuclease P1 were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized nuclease P1 were obtained in 0.2 M acetate buffer at pH 4.5 and a temperature of 70 °C. An increase in Km (from 3.165 to 18.125 mg mL−1) and a decrease in Vmax (from 1667.18 to 443.95 U min−1 mL−1) were recorded after immobilization. SPPSD-glutaraldehyde-nuclease P1 exhibited better thermal stability than the free enzyme. The apparent activation energy (Ea) of the free and immobilized nuclease P1 was 137.04 kJ mol−1 and 98.43 kJ mol−1, respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit.