title: Novel regenerative large-volume immobilized enzyme reactor: preparation, characterization and application.
abstract: A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion.
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China. Electronic address: guihuaruan@hotmail.com.
authors: Guihua
authors: G
authors: Ruan
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Meiping
authors: M
authors: Wei
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Zhengyi
authors: Z
authors: Chen
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Rihui
authors: R
authors: Su
authors: College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China.
authors: Fuyou
authors: F
authors: Du
authors: Shenzhen Academy of Metrology and Quality Inspection, Shenzhen, 518131, China.
authors: Yanjie
authors: Y
authors: Zheng
mesh_terms: D000818:Animals; D019149:Bioreactors; D002417:Cattle; D045304:Cytochromes c; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D017743:Equipment Reuse; D020441:Ginkgo biloba; D008956:Models, Chemical; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry*; D013552:Swine
chemical_list: D004800:Enzymes, Immobilized; D011506:Proteins; D012822:Silicon Dioxide; D045304:Cytochromes c
keywords: Enzyme immobilization; On-column digestion; Organic–inorganic hybrid silica particles; Regenerative enzyme reactor