Immobilization and purification of xylanase and β-glucanase by using the artificial oil body system
Date Issued
2008
Date
2008
Author(s)
Hung, Ying-Ching
Abstract
Xylanases and cellulases are the two major groups of industrial enzymes due to their fibrolytic functions and the potential applications to a big range of industrial processes. These enzymes are produced by immobilization or purification techniques. One of the commendable techniques is called artificial oil body system, also called AOB system. During the previous years, the AOB system had been established and has provided as a novel method for enzyme immobilization and purification. The objective of this study is to investigate the efficiency of immobilization and purification of the xylanase and β-glucanase by using the AOB system. The two target enzyme genes used in this study are xyn-CDBFV and 1,3-1,4-β-D-glucan 4- glucanohydrolase gene. The xyn-CDBFV is a xylanase gene from rumen fungus, Neocallimastix patriciarum, and the 1,3-1,4-β-D-glucan 4-glucanohydrolase gene is from rumen bacterium, Fibrobacter succinogenes. Both of the enzyme genes were first overexpressed in Escherichia coli as recombinant proteins fused to the N terminus of oleosin with intein as a linker. These two recombinant enzymes were then immobilized on AOB through the process of AOB reconstitution. Afterward, response surface methodology (RSM) was applied to identify the most optimal reaction condition of the AOB-immobilized xylanase and AOB-immobilized β-glucanase. As the results, the optimal reaction condition for the highest AOB-immobilized xylanase activity (3.93 U/mg of total protein) was observed at pH 6 and 59℃, whereas it for the highest AOB-immobilized β-glucanase activity (6.9 U/mg of total protein) was observed at pH 8.8 and 39℃. Additionally, AOB-immobilized xylanase retained 60% of its maximal activity after 120 minutes at 59℃; AOB-immobilized β-glucanase retain 67% of its maximal activity after 120 minutes at 40℃, but only retain 10% at 50℃. They could be recycled by brief centrifugation. After reusing nigh times, AOB-immobilized xylanase still retained more than 70% of the original activity; while the activity of AOB-immobilized β-glucanase dropped to 60% for reusing 10 times. In terms of purification, xylanase and β-glucanase could be released from AOB by inducing self-splicing of intein and centrifuging. In this study, the results show that AOB system is an applicable method on immobilizing and purifying recombinant rumen microbial xylanase and β-glucanase. Through the system, the two enzymes could then possibly be applied to industrial purpose.
Subjects
Artificial oil body
xylanase
β-glucanase
Type
thesis
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