Preparation of the immobilized metal ion membrane for enzyme immobilization: D-Hydantoinase and Candida antartica lipase B

• Main Authors: Yi-Miao Ko, 柯怡妙
• Other Authors: Yung-Chuan Liu
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/hhw876

博士 === 國立中興大學 === 化學工程學系所 === 101 === D-Hydantoinase (DHTase) is an important bioenzyme, which could hydrolysis D-5-substituted hydantoin to N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG). Then, an-other enzyme N-carbamoyl-D-amino acid amidohydrolase (DCase) would hydrolysis Nca-HPG to D-p-Hydroxyphenylglycine (D-p-HGP). D-p-HGP could react with 6-aminopenicillanic acid (6-APA) and become to Amoxicillin. Amoxicillin、Cefadroxil、Cefatrizine、Cefaparole and Cefaperazon areβ-lactam antibiotics. The global market demand of β-lactam antibiotics reaches thousands of tons annually for medical treatment. Lipases (EC 3.1.1.3) catalyze the hydrolysis of esters formed from glycerol and long-chain fatty acids. The activity of lipases is dramatically increased upon binding to the lipid surface, due to a conformational change of the enzyme. Candida antarctica lipase B (CalB) is preferred in many applications, because of its versatility with respect to substrates, high resistance to organic solvents, high thermal stability, stereo specificity and high enanti-oselectivity. Therefore, CalB is one of the wildly used biocatalysts in industry, including ki-netic resolutions, aminolysis, esterification, and transesterification. Among the recently developed purification techniques, immobilized metal ion affinity membrane (IMAM) has been widely applied in the enzyme purification processes with ad-vantages such as no intra-particle diffusion, short diffusion path, low pressure drop and easier scale up, which limited in conventional packed-column systems. Besides, How to adopt effi-cient immobilization technique to overcome some of the problems of enzymes as industrial biocatalysts: enzyme recovery, enzyme stability, reusiblity and storage are a critical focal point. A suitable immobilized method is the key point that greatly increases the possibilities of success. In this paper, it contains four parts. From part 1 to 3, how to prepare hydrophilic im-mobilized metal ion membrane for DHTase purification and immobilization is our object. In part 4, hydrophobic immobilized metal ion membrane has been prepared and immobilized CalB. In part 1, the complex effects of metal ions on DHTase purification with an immobi-lized metal affinity membrane was explored. Batch DHTase adsorption experiments showed that the adsorption capacity varied remarkably for IMAMs with different metal ions. The maximum adsorption of DHTase (1.513 ± 0.12 mg) was reached when using Cu2+ as the che-lated ion, whereas the Co2+ showed the highest activity on membrane with only small amounts of protein adsorption. The Mn2+, Co2+, Ni2+, Fe2+ and Fe3+ additions showed a posi- tive effect on DHTase activity. The addition of Cu2+ showed a varied effect from the inhibition on original DHTase to the promotion on Ni-purified DHTase. The purification folds using IMAM chelated with Co2+, Ni2+, and Zn2+ were in the range of six to seven. SDS-PAGE anal-ysis showed that all of the IMAM-purified DHTase exhibited the same molecular weight, meaning DHTase adsorbed on IMAM was highly specific. The DHTase purified by different metal ions showed various levels of increased activity when adding the corresponding metal ions. The addition of Mn2+ or Co2+ displayed a dramatic increase (9- to 10-fold) in activity of DHTase purified by IMAM chelated with the same ion. In part 2, this study constructs the IMAM via using chemical reagents and nickel ion on the regenerated cellulose membrane (RC membrane) to immobilized DHTase. The immobili-zation conditions were studied and the optimal conditions are as follows. By employing an IMAM with nickel ion of 155.5±5 μmol/disc immersed in 0.1 M Tris-HCl buffer pH 8 (with 0.8 M sodium chloride) and immobilized time of 14 h, a DHTase activity of 4.2±0.3 U/disc was obtained. The immobilized DHTase membrane can achieve a larger pH and thermal tol-erant range than that of free enzyme. Meanwhile, the stability test showed that 99% of en-zyme activity could be retained after being repeated 15-times. The storage test also displayed 99% enzyme preservation after 7 weeks of storage (0.1M, pH8, Tris-HCl buffer at 4 ℃). In part 3, various IMAMs were prepared from the RC membrane and chelated with various metal ions such as Co2+, Ni2+, Cu2+ and Zn2+. The adsorption isotherm and the kinetic parameters Vmax, Km of DHTase on IMAMs were studied. The Co-IMAM was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4±4.7 μmol/disc with a DHTase activity of 4.1±0.1 U/disc. Only 0.08 mg/disc protein was coupled onto Co-IMAM, but exhibited a similar activity as that us-ing Ni as ligand. Owing to this characteristic, a remarkably high specific activity of 51.9 U/mg was obtained for Co-IMAM, which is 4.9-fold higher than that of Ni-IMAM. The phe-nomenon of pH and thermal tolerant is similar to Ni-IMAM. The 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C in 0.1M, pH8 Tris-HCl buffer. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobi-lization operation. In part 4, the hydrophobic immobilized metal affinity membranes were constructed by using 3-Glycidoxypropyltrimethoxysilane (GPTMS) to form the meshed structure support. HCl was the best reagent used to open the GPTMS epoxy ring. However, higher concentration of HCl would distort the RC membrane and weaken its structure for further use. According to the optimization approach, the optimal reaction conditions were found as follows: a RC membrane immersed in 24 ml, 18.75％ HCl and 1 ml GPTMS under 24 °C, 150 rpm for 12 h. IDA was used as the chelating agent to bind nickel ions. In this case, the chelated ions and coupling CalB activity were significantly increased to 71.5±1.5μmol/disc and 6.74 ± 0.2U/disc respectively. Meanwhile, the stability test showed that 95% of enzyme activity could be retained after 10-times repeated use. The storage test also displayed 97% enzyme could be preserved after 4 weeks of storage.