School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia.
Int J Biol Macromol. 2020 May 1;150:80-89. doi: 10.1016/j.ijbiomac.2020.02.032. Epub 2020 Feb 5.
Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (K = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.
低聚麦芽糖（MOSs）是食品应用中新兴的低聚糖，可以通过从解淀粉芽孢杆菌 G1（Mag1）中酶法合成麦芽寡糖基转移酶来合成。然而，酶的稳定性不足使得这种方法在工业应用中不切实际。交联酶聚集体（CLEAs）的形成是提高酶稳定性的一种很有前途的方法，通过添加多孔剂来克服 CLEA 形成的底物可及性问题，从而生成多孔交联酶聚集体（p-CLEAs）。然而，p-CLEAs 表现出较高的酶浸出率和较低的溶剂耐受性。为了解决这些问题，将 Mag1 的 p-CLEAs（Mag1-p-CLEAs）包埋在海藻酸钠珠（CA）中。用 2.5%（w/v）海藻酸钠和 0.6%（w/v）氯化钙制备的 Mag1-p-CLEAs-CA 产生了 53.16%（17.0 U/mg）的活性，并且显示出比包埋的游离 Mag1（Mag1-CA）和包埋的非多孔 Mag1-CLEAs（Mag1-CLEAs-CA）更低的失活率和更长的半衰期。此外，与 Mag1-p-CLEAs 相比，Mag1-p-CLEAs-CA 在各种溶剂中表现出较低的酶浸出率和较高的耐受性。动力学研究表明，Mag1-p-CLEAs-CA 对β-环糊精（β-CD）具有相对较高的亲和力（K=0.62 mM）。在 50°C 下，Mag1-p-CLEAs-CA 合成了 300mg/g 的 MOSs。最后，Mag1-p-CLEAs-CA 的可重复使用性使它们成为 MOSs 连续合成的潜在生物催化剂。
