Elnashar Magdy M, Awad Ghada E, Hassan Mohamed E, Mohy Eldin Mohamed S, Haroun Bakry M, El-Diwany Ahmed I
Polymer Department, National Research Centre, El-Behouth Street, Dokki, Cairo 12311, Egypt ; Centre of Scientific Excellence, Group of Biopolymers and Nanobiotechnology, National Research Centre, El-Behouth Street, Dokki, Cairo 12311, Egypt.
Centre of Scientific Excellence, Group of Biopolymers and Nanobiotechnology, National Research Centre, El-Behouth Street, Dokki, Cairo 12311, Egypt ; Chemistry Natural & Microbial Products, National Research Centre, El-Behouth Street, Dokki, Cairo 12311, Egypt.
ScientificWorldJournal. 2014 Feb 2;2014:571682. doi: 10.1155/2014/571682. eCollection 2014.
β-Galactosidase (β-gal) was immobilized by covalent binding on novel κ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2(2) full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 °C compared to the free enzyme. The apparent K(m) after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity Vmax was 131.2 μ mol · min(-1) while it was 177.1 μ mol · min(-1) for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the κ-carrageenan beads loaded with β -galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.
通过两步法活化的新型κ-卡拉胶凝胶珠上，β-半乳糖苷酶（β-gal）通过共价结合固定化；凝胶珠先浸泡在聚乙烯亚胺中，然后浸泡在戊二醛中。采用2（2）全因子中心复合实验设计来优化最大酶负载效率的条件。通过将40单位的酶与凝胶珠浸泡8小时，实现了11.443 U酶/克凝胶珠的负载量。与游离酶相比，固定化过程使pH从4.5提高到5.5，操作温度从50℃提高到55℃。固定化后的表观K(m)为61.6 mM，而游离酶为22.9 mM。最大速度Vmax为131.2 μmol·min(-1)，而游离酶为177.1 μmol·min(-1)。全转化实验表明，固定化酶形式与游离酶一样具有活性，因为它们在4小时时都达到了最大100%的相对水解率。可重复使用性测试证明，负载β-半乳糖苷酶的κ-卡拉胶珠可耐用20个循环，保留60%的固定化酶活性，更便于工业应用。
