Preparation and characterization of -amylase-immobilized thermal-responsive composite hydrogel membranes Yi-Ming Sun, 1 Jyh-Ping Chen, 2 Ding-Hsin Chu 1 1 Department of Chemical Engineering, Yuan Ze University, Chung-Li, Taoyuan, Taiwan 320, Republic of China 2 Department of Chemical Engineering, Chang Gung University, Kwei-San, Taoyuan, Taiwan 333, Republic of China Received 29 September 1997; accepted 5 October 1998 Abstract: Composite hydrogel membranes of crosslinked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co- 2-hydroxyethyl methacrylate) [P(NIPAAm-NAS-HEMA)] with starch, as a macropore forming agent, on nonwoven polyester was prepared. The membranes could swell and de-swell around the characteristic lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAAm). It was demonstrated that the presence of mac- ropores in the membranes could improve the immobiliza- tion efficiency as well as lead to a short responding time upon temperature change across the LCST. Immobilized -amylase could retain as high as 33% of the activity of the free enzyme with a loading level of 0.60–0.65 mg/cm 2 when the membrane preparation and enzyme immobilization con- ditions were optimized. The half time (T 0.5 ) for the swelling or de-swelling response of the gel phase within the mem- branes was less than 2 min, and the 90% time (T 0.9 ) was less than 6 min. The permeability for maltose through the mem- branes could change as much as 4.9-fold when the tempera- ture was raised above or reduced below the LCST. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 45, 125–132, 1999. Key words: poly(N-isopropylacrylamide); -amylase; tem- perature-sensitive hydrogel; enzyme immobilization; swell- ing kinetics INTRODUCTION It is well known that poly(N-isopropylacrylamide) (PNIPAAm) in aqueous solution has a lower critical solution temperature (LCST), around 32°–33°C. 1–3 The polymer in linear or branch form dissolves in water below the LCST and precipitates above it. When the polymer is crosslinked, it forms a thermal-responsive hydrogel, which swells below and de-swells above the LCST. The reversible thermal-responsive behavior of this polymer or its gels has been utilized in many biomedical applications, such as controlled drug release, 4–6 protein purification and affinity separa- tion, 7–10 immobilization of enzymes or bioactive ma- terials, 11–18 and biosensing. 19,20 However, a conven- tional hydrogel of PNIPAAm or its copolymers me- chanically is weak and fragile. There is a need to make the hydrogel mechanically stronger if it is to be used in a severe biological environment. In one previous report 13 from our group, linear PNIPAAm with an end-capped carboxyl group and a linear copolymer of NIPAAm and N-acryloxysuccin- imide (NAS) were synthesized. The end-capped car- boxyl group of the former was activated by esterifica- tion with N-hydroxysuccinimide (NHS), and the suc- cinimide groups in both polymers could be used to conjugate an enzyme molecule. 14,15 The polymer– enzyme conjugates were soluble below and precipi- tated above the LCST, and this reversible dissolution– precipitation property enabled enzyme recovery from a reaction solution after the enzyme reaction was com- pleted. In the current study, composite hydrogel mem- branes made of crosslinked poly(N-isopropylacryl- amide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) [P(NIPAAm-NAS-HEMA)] on non- woven polyester have been synthesized. The compos- ite hydrogel membranes provide the succinimide groups for enzyme immobilization and temperature- sensitive swelling–de-swelling properties, and they are as strong mechanically as the nonwoven original. Usually, the immobilized enzyme on a solid support has better recovery but encounters problems of sig- nificant mass transfer resistance. In our method, soluble starch has been added in the formulation. The Correspondence to: Y.-M. Sun; e-mail: cesunym@saturn. yzu.edu.tw Contract grant sponsor: National Science Council, Repub- lic of China; contract grant number: NSC-86-2214-E-182-007 © 1999 John Wiley & Sons, Inc. CCC 0021-9304/99/020125-08