[논문]Cadaverine Production by Using Cross-Linked Enzyme Aggregate of Escherichia coli Lysine Decarboxylase

Park, Se Hyeon; Soetyono, Feilicia; Kim, Hyung Kwoun

doi:10.4014/jmb.1608.08033

Cadaverine Production by Using Cross-Linked Enzyme Aggregate of Escherichia coli Lysine Decarboxylase 원문보기

Journal of microbiology and biotechnology, v.27 no.2, 2017년, pp.289 - 296

Park, Se Hyeon (Division of Biotechnology The Catholic University of Korea) , Soetyono, Feilicia (Division of Biotechnology The Catholic University of Korea) , Kim, Hyung Kwoun (Division of Biotechnology The Catholic University of Korea)

Abstract ▼ AI-Helper

Lysine decarboxylase (CadA) converts ${\small{L}}-lysine$ into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using $CadA^{CLEA}$. $CadA^{free}$ and $CadA^{CLEA}$ were characterized for their enzymatic properties. The optimum temperatures of $CadA^{free}$ and $CadA^{CLEA}$ were $60^{\circ}C$ and $55^{\circ}C$, respectively. The thermostability of $CadA^{CLEA}$ was significantly higher than that of $CadA^{free}$. The optimum pH of both enzymes was 6.0. $CadA^{free}$ could not be recovered after use, whereas $CadA^{CLEA}$ was rapidly recovered and the residual activity was 53% after the $10^{th}$ recycle. These results demonstrate that $CadA^{CLEA}$ can be used as a potential catalyst for efficient production of cadaverine.

주제어

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제안 방법

A recovery test was performed using CadA^CLEA. After CadA^CLEA was used in the first reaction, it was recovered by centrifugation at 10,000 ×g for 5 min.
In order to determine the optimum temperature and pH, the activity of CadA^free and CadA^CLEA were evaluated at various temperatures and initial pH values (Figs. 3A and 3B). Both CadA^free and CadA^CLEA showed similar relative activity at pH 5-8.
Preparation of CLEAs is a useful method for the immobilization of industrial enzymes. In this research, CadA^CLEA was prepared by ammonium sulfate precipitation and a subsequent glutaraldehyde cross-linking process. The activity of CadA^CLEA was 31.
In this study, we prepared a CLEA using a cell-free extract of an E. coli strain overexpressing the cadA gene, characterized its biochemical properties, and compared CadA^CLEA with CadA^free enzyme. Subsequently, we performed bioconversion using CadA^CLEA for cadaverine production (Scheme 1).
The effects of temperature and pH were evaluated using CadA^free and CadA^CLEA. Their enzyme activities were assayed at various temperatures (20-70o C) to determine the optimum temperature for lysine decarboxylation.

대상 데이터

Primer sequences used were as follows: CadAforward (5’-GATCATATGAACGTTATTGCAATA-3’) and CadAreverse (5’-GATAAGCTTTTTTTTGCTTTCTTCTTTCAA-3’).

이론/모형

a Protein concentration of soluble CadA and CadA^CLEA were measured by the Bradford method.

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