Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes.
cephalosporin C acylase
enzyme immobilization
pHluorin
protein distribution
shaking speed
Journal
Biotechnology progress
ISSN: 1520-6033
Titre abrégé: Biotechnol Prog
Pays: United States
ID NLM: 8506292
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
14
03
2020
revised:
31
07
2020
accepted:
05
08
2020
pubmed:
11
8
2020
medline:
29
1
2022
entrez:
11
8
2020
Statut:
ppublish
Résumé
During enzyme immobilization, enzyme activity and protein distribution are affected by various factors such as enzyme load, temperature, and pH. In general, two types of protein distribution patterns (heterogeneous or homogeneous) are observed inside a porous carrier, owing to differences in preparation parameters. During the immobilization of a fusion protein (CCApH) of cephalosporin C acylase (CCA) and pHluorin (a pH-sensitive mutant of green fluorescent protein), different shaking speeds induced obvious differences in protein distribution on an epoxy carrier, LX-1000EPC. Enzyme immobilization with a homogeneous distribution pattern was observed at a low shaking speed (120 rpm) with an operational stability of 10 batches at 37°C. The operational stability of an immobilisate with heterogeneous protein distribution prepared at a high shaking speed (200 rpm) was six batches. Given the pH-sensitive characteristics of pHluorin in the fusion protein, the intraparticle pH of CCApH immobilisates during catalysis was monitored using confocal laser scanning microscopy. The microenvironmental pH of the immobilisate with heterogeneous protein distribution sharply decreased by about 2 units; this decrease in the pH may be detrimental to the life-span of immobilized CCA. Thus, this work demonstrates the good operational stability of pH-sensitive proton-forming immobilized enzymes with homogeneous protein distribution.
Substances chimiques
Enzymes, Immobilized
0
Escherichia coli Proteins
0
Recombinant Fusion Proteins
0
Green Fluorescent Proteins
147336-22-9
Penicillin Amidase
EC 3.5.1.11
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e3063Subventions
Organisme : National Natural Science Foundation of China
ID : 21476025
Informations de copyright
© 2020 American Institute of Chemical Engineers.
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