Instituto de Investigaciones para la Industria Química (INIQUI), Universidad Nacional de Salta-CONICET, Buenos Aires, 177-4400 Salta, Argentina.
Appl Microbiol Biotechnol. 2012 Feb;93(3):1127-34. doi: 10.1007/s00253-011-3469-y. Epub 2011 Jul 22.
α-L-rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity-pH and activity-temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40-70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K (m) of α-L-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).
从土曲霉中提取的α-L-鼠李糖苷酶通过共价键固定在以下磁性载体上：聚对苯二甲酸乙二醇酯（丹宁酰肼）、聚硅氧烷/聚乙烯醇（POS/PVA）和壳聚糖。通过使用氯化铁和氯化亚铁的热共沉淀法对粉状载体进行磁化，并用戊二醛进行固定化。丹宁酰肼（0.53 nkat/μg 蛋白质）和 POS/PVA（0.59 nkat/μg 蛋白质）固定化酶的活性明显高于壳聚糖衍生物（0.06 nkat/μg 蛋白质）。与游离酶相比，所有固定化酶的活性-pH 和活性-温度曲线没有差异，除了壳聚糖衍生物在 65°C 时表现出更高的最高温度。丹宁酰肼衍生物的热稳定性在 40-70°C 的温度范围内表现出与游离酶相似的行为。POS/PVA 和壳聚糖衍生物在 60°C 以下稳定，但在 70°C 时完全失活。经过十次连续重复使用后，制剂的活性没有明显下降。固定在磁化丹宁酰肼上的α-L-鼠李糖苷酶（1.05±0.22 mM）、POS/PVA（0.57±0.09 mM）和壳聚糖（1.78±0.24 mM）的表观 K（m）值均高于估计的游离酶（0.30±0.03 mM）。与游离酶相比，丹宁酰肼酶衍生物在水解 0.3%narigin（分别在 1 小时后为 91%和 73%）和合成鼠李糖苷（分别在 1 小时后为 0.116 和 0.014 mg 芦丁）方面表现更好。
