Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Lactulose Production Using Immobilized Cells Including Thermostable Cellobiose 2-epimerase

열내성 Cellobiose 2-epimerase를 발현하는 대장균의 고정화담체를 이용한 락툴로오스의 생산방법

  • 박아름 ((주)포바이오코리아 기술개발연구소) ;
  • 구봉성 ((주)포바이오코리아 기술개발연구소) ;
  • 김진숙 ((주)포바이오코리아 기술개발연구소) ;
  • 김은정 ((주)포바이오코리아 기술개발연구소) ;
  • 이현철 ((주)포바이오코리아 기술개발연구소)
  • Received : 2016.09.12
  • Accepted : 2016.10.06
  • Published : 2016.12.28
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Abstract

Lactulose, a synthetic disaccharide, has received increasing interest because of its role as a prebiotic that can increase the proliferation of Bifidobacterium and Lactobacillus spp. and enhance the absorption of calcium and magnesium. While the industrial production of lactulose is still mainly achieved by the chemical isomerization of lactose in alkaline media, this process has drawbacks including the need to remove catalysts and by-products, as well as high energy requirements. Recently, the use of cellobiose 2-epimerase (CE) has been considered an interesting alternative for industrial lactulose production. In this study, to develop a process for enzymatic lactulose production using CE, we screened improved mutant enzymes ($CS-H^RC^E$) from a library generated by an error-prone PCR technique. The thermostability of one mutant was enhanced, conferring stability up to $75^{\circ}C$, and its lactulose conversion yield was increased by 1.3-fold compared with that of wild-type CE. Using a recombinant Escherichia coli strain harboring a CS35 $H^RC^E$-expressing plasmid, we prepared cell beads immobilized on a Ca-alginate substrate and optimized their reaction conditions. In a batch reaction with 200 g/l lactose solution and the immobilized cell beads, lactose was converted into lactulose with a conversion yield of 43% in 2 h. In a repeated 38-plex batch reaction, the immobilized cell beads were relatively stable, and 80% of the original enzyme activity was retained after 4 cycles. In conclusion, we developed a reasonable method for lactulose production by immobilizing cells expressing thermostable CE. Further development is required to apply this approach at an industrial scale.

락툴로오스는 기존에 화학적인 이성화법을 통해 생산해왔던 기능성 당으로서 프로바이오틱스나 장내균총 개선을 위한 의약품으로 활용되어 왔다. 최근 락툴로오스 화학전환법의 단점인 촉매제거와 부산물제거 에너지손실등의 문제를 해결할 수 있는 생물촉매를 이용한 락툴로오스 전환법이 대두되었다. 본연구에서는 유당의 낮은 용해도와 락툴로오스의 효율적전환을 위해 최적의 효소를 선별하여 무작위 돌연변이법으로 유전자를 개량하여 열내성이 $75^{\circ}C$까지 증진되고 활성이 1.3배 향상된 효소를 선별하였다. 이 효소를 정제하여 사용하는 대신 본 연구에서는 과량 발현시킨 대장균을 Ca-alginate로 고정화하여 $70^{\circ}C$에서 200 g/l의 유당과 회분식으로 반응시켜 43%의 전환 수율을 확인하였다. 반복회분식 실험에서 고정화된 담체는 비교적 안정적이었으며 4회 반복반응 후에도 80% 이상의 활성을 유지하고 있었다. 산업적인 방법을 개발하기 위해 고정화 담체를 이용한 반응기의 운전 최적화와 담체의 안정화를 증진시키는 추가적인 연구가 필요하지만, 본 연구에서는 열내성 특성을 이용하여 정제된 효소가 아닌 효소를 발현하는 세포자체를 고정화 시킴으로써 경제성있는 생산에 대한 방법론을 제시하였다.

Keywords

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