静电纺丝制备苯乙烯马来酸酐共聚物纳米纤维及其固定化β-D-半乳糖苷酶

Preparation of Electrospun Poly(styrene-co-maleic anhydride) Nanofibers and Their Use in Immobilization of β-D-Galactosidase

采用静电纺丝技术制备苯乙烯-马来酸酐共聚物纳米纤维,最佳电纺条件为:聚合物浓度0.35g/mL、针尖到接收板距离25cm、电纺液流量250μL/h、电压21kV.该条件下获得了直径约300nm且分布均一的纳米纤维.利用该纳米纤维固定β-D-半乳糖苷酶,固定化反应的最适pH值为4.0,此时酶负载量为(15.1±0.5)mg/g.固定化酶催化2-硝基苯酚-β-D-半乳吡喃糖苷水解反应的米氏常数K_m=2.7mmol/L,略大于游离酶的K_m值(2.2mmol/L);最大反应速率V_(max)为97.2μmol/(min·mg),为游离酶的47.8%.固定化酶在37℃下重复操作21次后活性损失仅为15%.在连续搅拌式反应器中将固定化酶用于催化乳糖的水解反应,连续使用17d仍能稳定运行.

Unique poly（styrene-co-maleic anhydride） nanofibers with a uniform diameter of about 300 nm were prepared by eletrospinning a solution containing 0.35 g/mL polymer,25 cm gap between ejection needle tip and collector,flow rate of 250 L/h,and applied voltage of 21 kV.The electrospun nanofibers were applied subsequently for immobilization of β-D-galactosidase,an enzyme loading of（15.1±0.5） mg protein/g membrane was obtained.The immobilized enzyme demonstrated a Michealis constant（K_m） of 2.7 mmol/L,slightly higher than that of the native enzyme（2.2 mmol/L）.The maximum reaction velocity（V_（max）） of the immobilized enzyme was found as 97.2 mol/（min·mg）,47.8% that of native enzyme.The immobilized enzyme showed an excellent reusability such that 21 reusing cycles only led to 15% loss of the enzyme activity.Furthermore,the immobilized β-D-galactosidase was suitable for hydrolysis of lactose in a continuous stirring tank reactor that was operated steadily for 17 d.