꿀벌(Apis mellifera L.) 일벌독의 생체아민 cadaverine 함량 및 분석법

최홍민(Hong Min Choi),김효영(Hyo Young Kim),김세건(Se Gun Kim),한상미(Sang Mi Han) 한국생명과학회 2021 생명과학회지 Vol.31 No.2

본 연구의 목적은 항염, 항균 등 다양한 기능성을 갖는 서양종꿀벌(Apis mellifera L.)의 일벌독에서 분리한 봉독의 활성아민 성분과 함량을 분석하고 그 분석법을 검토하였다. 본 연구에서는 Ultra Performance Liquid Chromatography에 Halo C18컬럼과, acetonitrile 및 증류수를 이동상으로 사용하여 분석시간 13분 이내에서 봉독의 cadaverine에 대한 성분 분석법을 개발하였고, 분석법 밸리데이션을 통하여 특이성, 정확성, 정밀성을 갖춘 분석법임이 확인되었다. 봉독의 cadaverine의 직선성은 R²=0.99 이상으로 측정되어 양호하게 분석되었고, 검출한계는 0.3 ㎍/ml, 정량한계는 0.3 ㎍/ml으로 나타났으며, 회수율은 97.6~99.1%로 나타났다. Cadaverine의 일내 정밀도는 상대표준편차(RSD) 값이 0.25~0.44%였으며, 일간 정밀도는 0.25~1.25%로 상대표준편차 값이 5% 이내의 우수한 정밀성을 갖는 것으로 확인되었다. 이러한 분석법을 통해 지금까지 봉독에서는 보고된 바 없는 활성아민 cadaverine이 평균 1.10±0.05 ㎎/g 함유된 것으로 확인되었다. 따라서 본 연구는 봉독에서 cadaverine 함량과 cadaverine 분석법에 대한 직선성, 검출한계, 정량한계 및 회수율을 측정한 결과, 모두 만족스러운 결과를 나타냈으며, 봉독의 cadaverine에 대한 기초자료와 생리활성에 연구로 사용될 수 있을 것으로 기대된다. This study aimed to analyze the content and composition of a biological amine, cadaverine, isolated from the venom of worker honeybees (Apis mellifera L.). This biological amine-which has diverse functionality, such as anti-inflammatory and antibacterial effects-has not been previously reported in bee venom. An assay completed in 13 minutes was developed for the cadaverine present in the bee venom using an ultra-performance liquid chromatograph and a Halo C18 column with acetonitrile and water as the mobile phase. The specificity, accuracy, and precision of the assay were verified, and the assay was validated. The linearity for cadaverine in the bee venom was R²=0.99 or above, indicating a moderate level. The limit of detection and limit of quantification were both 0.3 ㎍/ml, and the rate of recovery was 97.6%-99.1%. The relative standard deviation (RSD) of the intra-day precision and inter-day precision for cadaverine was 0.25%-0.44% and 0.25%-1.25%, respectively, with an RSD that fell within 5% indicating excellent precision. Through this novel assay, it was found that the mean content of cadaverine was 1.10±0.05 ㎎/g. Our results indicated that the linearity, limit of detection, limit of quantification, and rate of recovery of the cadaverine assay were of a satisfactory level, and the cadaverine content of the bee venom was ably determined. This study provides basic data on cadaverine in bee venom, which will prove useful in further studies on the bioactivity of this component.

Directed Evolution and Mutagenesis of Lysine Decarboxylase from Hafnia alvei AS1.1009 to Improve Its Activity toward Efficient Cadaverine Production

Chen Wang,Kai Zhang,Chen Zhongjun,Heng Cai,Wan Honggui,Ping-Kai Ouyang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3

Lysine decarboxylase (LDC) exhibits a significant role in cadaverine (1,5-pentanediamine, diaminopentane) production from lysine. In this study, an error-prone PCR and DNA shuffling were performed to improve the activity of LDC from Hafnia alvei AS1.1009 for cadaverine production. A sensitive high-throughput screening strategy based on a pH indicator was established for directed evolution of LDC. Several improved mutants were obtained from directed evolution and LDCV147F/E583G mutant showed highest activity to catalyze lysine to cadaverine. This mutant showed 1.62-fold high LDC activity when compared to wild-type. Further analysis by site-directed mutagenesis reveled that only the mutant E583G was sufficient for higher catalytic activity. Wild type LDC and mutant LDCE583G were purified by an improved method including hydrophobic chromatography. These purified enzymes were characterized and the kinetic parameters were compared between LDCE583G and WT LDC. Vmax of LDCE583G was 1.32-fold higher than that of WT LDC. Use of LDCE583G mutant showed 1.48-fold improved productivity of cadaverine when compared to wild type. The concentration of cadaverine in E. coli JM109/pTrc99a-ldc2-41 was 63.9 g/L with conversion yield of 93.4% during 5 h. These results indicate that the mutation has positive effects on improving LDC activity and a potential candidate for cadaverine production.

Development of a continuous L-lysine bioconversion system for cadaverine production

김정호,서형민,가네산,Shashi Kant Bhatia,송헌석,김준영,전종민,윤정준,김윤곤,박경문,양영훈 한국공업화학회 2017 Journal of Industrial and Engineering Chemistry Vol.46 No.-

Cadaverine, afive carbon diamine (1,5-diaminopentane), plays a role as a building block of polyamidesand it can be made by fermentation or direct bioconversion. To improve its production by increasingreusability of immobilized enzyme and avoid separation of enzyme in bioconversion, a continuousL-lysine bioconversion process for cadaverine production has been developed. Various divalent cations,alginate concentrations, cell density with alginate andflow rate of feed were examined to maximize thelysine decarboxylase activity of the whole-cell immobilized beads. Under the selected conditions,123 h ofcontinuous cadaverine production has been performed and 5.5 L of 819 mM cadaverine were producedwith 14 mL reactor resulting in 466.5 g of cadaverine. Cadaverine production was possible with smallvolume reactor maintaining relatively high concentration of substrate

Development of a continuous l-lysine bioconversion system for cadaverine production

Kim, J.H.,Seo, H.M.,Sathiyanarayanan, G.,Bhatia, S.K.,Song, H.S.,Kim, J.,Jeon, J.M.,Yoon, J.J.,Kim, Y.G.,Park, K.,Yang, Y.H. Korean Society of Industrial and Engineering Chemi 2017 Journal of industrial and engineering chemistry Vol.46 No.-

<P>Cadaverine, a five carbon diamine (1,5-diaminopentane), plays a role as a building block of polyamides and it can be made by fermentation or direct bioconversion. To improve its production by increasing reusability of immobilized enzyme and avoid separation of enzyme in bioconversion, a continuous l-lysine bioconversion process for cadaverine production has been developed. Various divalent cations, alginate concentrations, cell density with alginate and flow rate of feed were examined to maximize the lysine decarboxylase activity of the whole-cell immobilized beads. Under the selected conditions, 123 h of continuous cadaverine production has been performed and 5.5 L of 819 mM cadaverine were produced with 14 mL reactor resulting in 466.5 g of cadaverine. Cadaverine production was possible with small volume reactor maintaining relatively high concentration of substrate. (C) 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.</P>

Cadaverine production from galactose in E. coli by redesigning metabolic pathway

백동엽,임현규,서상우,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

Biological production of cadaverine has been suggested as an alternative route to replace polyamides generated by the petroleum-based process. As most microorganisms cannot efficiently metabolize other biomass-derived sugars as fast as glucose, redesigning microorganisms is necessary for utilizing those carbon sources with enhanced carbon flux and product formation. In this study, we engineered E. coli to produce cadaverine with rapid assimilation of galactose, a promising future feedstock. To achieve this, genes related to the metabolic pathway were maximally expressed to amplify the flux toward cadaverine. The feedback inhibition of metabolic enzymes and degradation/re-uptake pathways was also inactivated to robustly produce cadaverine. The final strain DHK4 produced 8.80 g/L cadaverine with high yield and productivity during fed-batch fermentation, which was similar to or better than the previous glucose fermentation. This is the first report to produce cadaverine from galactose.

Development of cadaverine production from galactose in Escherichia coli

이건민,곽동훈,임현규,양진아,서상우,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.0

In this study, we engineered Escherichia coli to produce cadaverine with rapid assimilation of galactose, a promising future feedstock. To achieve this, genes related to the metabolic pathway were maximally expressed to amplify the flux toward cadaverine production via synthetic expression cassettes consisting of predictive and quantitative genetic parts (promoters, 5′-untranslated regions, and terminators). Furthermore, the feedback inhibition of metabolic enzymes and degradation/re-uptake pathways was inactivated to robustly produce cadaverine. Finally, the resultant strain, DHK4, produced 8.80 g/L cadaverine with high yield (0.170 g/g) and productivity (0.293 g/L/h) during fed-batch fermentation, which was similar to or better than the previous glucose fermentation. This is the first report to produce cadaverine from galactose. Moreover, the yield (0.170 g/g) was the highest among engineered E. coli systems.

Conversion of lysine into cadaverine by whole cell biocatalysts using Escherichia coli ldcC

신지현,김현중,김정호,이주희,송헌석,양영헌,박경문 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.0

1,5-diaminopentane(cadaverine) is a valuable chemical because of its wide industrial applications. Since there are not many reports of the direct production using biocatalysts, the enzymatic process using lysine decarboxylases(LDCs) was developed for cadaverine production from renewable material such as lysine. There are two well-known LDCs in Escherichia coli, cadA and ldcC. Among them, cadA is more popular than ldcC because cadA has two times higher biological activity in comparison with ldcC. In this study, lysine was converted into cadaverine by whole cell biocatalysts using E.coli BL21(DE3) expressing E.coli ldcC. In order to overcome the low expression level and the low conversion rate of ldcC, the effects of IPTG concentration and conversion temperature and pH were studied to increase the expression of ldcC and the conversion rate of lysine into cadaverine. Higher expression level of ldcC was observed, and 95% of lysine was consumed for conversion into cadaverine.

Selective recovery of cadaverine from lysine decarboxylase bioconversion solution using methyl ethyl ketone

홍윤기,김현중,전종민,문유미,홍주원,주정찬,송봉근,박경문,이상현,양영헌 한국공업화학회 2018 Journal of Industrial and Engineering Chemistry Vol.64 No.-

Efficient extraction and purification process is still a major bottleneck for economical production of cadaverine. We examined 10 different organic solvents to determine which one might be a suitable solvent for cadaverine extraction. Methyl ethyl ketone (MEK) was a very selective solvent for cadaverine. High pH was critical factor for cadaverine extraction with high purity. Cadaverine extraction efficiency of more than 70.1% with 99% of purity was successfully achieved by applying optimized extraction condition (pH 13.5, 58 °C, 200 rpm incubation for 6 h). Cadaverine extraction using MEK seems quite feasible and promising for the preparation of polyamide monomers for environmental process.

Metabolic engineering of <i>Escherichia coli</i> for the production of cadaverine: A five carbon diamine

Qian, Zhi‐,Gang,Xia, Xiao‐,Xia,Lee, Sang Yup Wiley Subscription Services, Inc., A Wiley Company 2011 Biotechnology and bioengineering Vol.108 No.1

<P><B>Abstract</B></P><P>A five carbon linear chain diamine, cadaverine (1,5‐diaminopentane), is an important platform chemical having many applications in chemical industry. Bio‐based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum‐based chemical synthesis. Here, we report development of a metabolically engineered strain of <I>Escherichia coli</I> that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, <SMALL>L</SMALL>‐lysine decarboxylase, which converts <SMALL>L</SMALL>‐lysine directly to cadaverine, was amplified by plasmid‐based overexpression of the <I>cadA</I> gene under the strong <I>tac</I> promoter. Furthermore, the <SMALL>L</SMALL>‐lysine biosynthetic pool was increased by the overexpression of the <I>dapA</I> gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong <I>trc</I> promoter in the genome. The final engineered strain was able to produce 9.61 g L<SUP>−1</SUP> of cadaverine with a productivity of 0.32 g L<SUP>−1</SUP> h<SUP>−1</SUP> by fed‐batch cultivation. The strategy reported here should be useful for the bio‐based production of cadaverine from renewable resources. Biotechnol. Bioeng. 2011; 108:93–103. © 2010 Wiley Periodicals, Inc.</P>

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

( Se Hyeon Park ),( Feilicia Soetyono ),( Hyung Kwoun Kim ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2

Lysine decarboxylase (CadA) converts 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 60oC and 55℃, 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 Cad^ACLEA 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.