Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/41162
標題: Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents
作者: Ho, L.F.
林松池
Li, S.Y.
Lin, S.C.
Wen-Hwei, H.
關鍵字: protein purification
enzyme immobilization
IMAC
recombinant escherichia-coli
containing cytochrome-c
d-hydantoinase
activity
ion affinity
industrial-production
fine chemicals
chromatography
proteins
acid
biocatalysis
期刊/報告no:: Process Biochemistry, Volume 39, Issue 11, Page(s) 1573-1581.
摘要: Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged D-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 mumol Cu2+/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of D,L-p-hydroxyphenylhydantoin (D,L-HPH) to N-carbamoyl-D-hydroxyphenylglycine, an intermediate for the production of D-hydroxylphenylglycine. Under optimal conditions, 60 degreesC and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation. (C) 2003 Elsevier Ltd. All rights reserved.
URI: http://hdl.handle.net/11455/41162
ISSN: 0032-9592
文章連結: http://dx.doi.org/10.1016/s0032-9592(03000288-7
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