Xylan을 분해하는 신규 미생물의 탐색 및 재조합 xylanase와 acetyl xylan esterase의 특성분석

Abstract

Xylan은 주로 식물의 세포벽 주성분 중 하나인 hemicellulose에 포함되는 다당류로서, 이를 분해하는 효소는 바이오에탄올 생산 당화공정, 목재 펄프 공정, 직물공정, 동물사료 가공 등의 다양한 산업에 이용되어진다. 본 연구에서는 xylan을 분해하는 신규 미생물들을 탐색하고 이들이 생산하는 효소를 E. coli로부터 재조합단백질로 생산하여 그 효소적 특성을 분석하고자 하였다. 기존 xylan 분해 미생물의 탐색은 주로 한천배지 상에서 staining을 통해 clear zone을 확인하는 방식으로 이루어져 왔으나 본 연구에서는 3,5-dinitrosalicyclic acid(DNS) 방법을 응용하여 96 microwell-plate상에서 대량으로 빠르게 탐색하였다. 먼저, xylan 분해 미생물을 찾기 위해 xylan 기질을 함유하고 있는 식물이나 해조류, 이를 섭식하는 동물의 내장 등에서 샘플을 채집하였다. 미생물을 탐색하기 위한 초기배지는 육상샘플과 해양샘플 배양용으로 구분하여 제작하였고, xylan을 단일 탄소원으로 첨가하고 지하수와 해수를 이용해 배지를 제작하였다. 배양된 미생물들은 초기배지에 yeast extract가 0.1%로 첨가된 액체배지 상에서 배양하여 그 상등액의 xylan 분해 활성을 확인함으로써 xylan 분해 미생물을 선별하였다. 전체 44개의 xylan 분해 미생물이 탐색되었고, 분해 활성을 나타내는 미생물들은 유전학적 동정을 통해 계통을 확인하였다. 그 결과 29종이 xylan 분해 미생물로 본 연구에서 처음 보고되었다. 이들 중 2개의 미생물(Streptomyces padanus J103, Ochrovirga pacifica S85)을 선별해 그 유전체를 분석하였다. S. padanus J103 유전체로부터 xylan 분해관련 유전자원들을 9개가 확인되었고, O. pacifica S85 유전체로부터는 2개의 유전자가 확인되었다. 각 xylan 분해관련 유전자원들은 유전자 진단 프로그램들을 이용해 신호서열, domain, active site 등을 확인하였다. 이들 유전자들을 활용해 E. coli로부터 재조합 단백질로 생산하기 위해서 S. padanus J103 유래 xylanase 유전자 2개(J103-x2, J103-x7)와 O. pacifica S85 유래 acetyl xylan esterase 유전자 1개(S85-x1)를 선별하였다. 선별된 유전자들은 pET expression vector 시스템을 이용해 cloning 하고 E. coli BL21(DE3) cell 안으로 형질전환 하여 단백질 발현을 유도하였다. E. coli로부터 생산된 재조합 단백질들(J103-x2, J103-x7, S85-x1)은 이온교환크로마토그래피를 이용해 정제하고 효소적 특성을 분석하였다. 정제된 두 xylanase(J103-x2, J103-x7)의 최적 pH와 최적 온도에 대한 효소활성을 측정한 결과, 두 xylanase 모두 pH 4.0 그리고 60°C에서 최적 활성을 나타내었다. 열안정성 측정을 통해 J103-x2는 50°C 조건 하에서 120분간 반응을 한 경우에 온도에 전반적으로 활성이 유지되었으나, J103-x7은 30분이 지나자 모든 효소활성이 사라졌다. Acetyl xylan esterase 재조합단백질인 S85-x1이 기존의 판매되는 xylanase(Megazyme, Aspergillus niger)와 비교하였을 때 시너지 효과가 나타나는지에 대하여 확인한 결과 정제된 acetyl xylan esterase는 단일로 xylan 기질에 반응하였을 때 활성을 전혀 가지지 않았고, 상용화된 xylanase와 복합 사용시 120분 후 xylanase만 사용한 것보다 beechwood xylan에 대한 활성이 약 2.1배 증가하는 것으로 나타났다. 이로써 acetyl xylan esterase 재조합단백질인 S85-x1이 xylanase와 함께 처리하였을 때 시너지 효과가 뛰어나게 나타나는 것을 확인하였다.|Xylan, a component of plant cell wall, is the most abundant non-cellulosic polysaccharide (hemicellulose) in nature. Xylanases can be use to digest xylan that has numerous use for several industries such as bioethanol production, bio-bleaching of pulp, textile, production of animal feed. In this study, we isolated many xylan-degrading microbes that were previously uncharacterized. The xylanase and acetyl xylan esterase were cloned and overexpressed in E. coli expression system, then the biochemical properties of purified recombinant enzymes were analyzed. Congo red staining method display clearing zone around xylanase positive colonies on agar plate, and this method is commonly used for investigating xylan-hydrolyzing activity. However, in this study, we adjusted microwell plate based on 3,5-dinitrosalicylic acid (DNS) method for screening xylan-degrading microbe. Decomposed xylan-containing plant and seaweed, and plant- or seaweed-consuming marine animal's intestines were sampled and screened for xylan-degrading microbes. Initial culture media were made with xylan as the sole carbon source with ground water or seawater. Subsequently isolated colonies were sub-cultured in broth containing yeast extract and xylan. Forty-four microbes that exhibit xylan hydrolyzing activity were determined by using our modified DNS method. These microbes were genetically identified, and 29 species were first reported as xylan-degrading microbes. Two strains (O. pacifica strain S85 and S. padanus strain J103) were selected for genome analysis from this pool. Whole genome sequencing was performed with GS-FLX 454 titanium. The eleven xylan-degrading relative enzymes (nine and two genes from S. padanus J103 and O. pacifica S85, respectively) were predicted by NCBI Blast algorithm. Signal peptides, domains and active sites of each genes were predicted by using web based prediction server. Two xylanase genes (J103-x2, J103-x7) from S. padanus J103 and one acetyl xylan esterase gene (S85-x1) from O. pacifica S85 were cloned and overexpressed in E. coli expression system. The recombinant enzymes were purified using ion-exchange chromatography with Ni+ charged column. Biochemical properties of purified recombinant enzymes were analyzed to establish optimum conditions. Both recombinant xylanases (J103-x2 and J103-x7) showed the highest activities at 60°C and pH 4.0. Although their optimum temperature and pH were almost similar, their thermostability showed a significant difference. The J103-x2 was stable and retaining more than 90% of its activity after pre-incubation at 50°C for 120 min, whereas J103-x7 lost most of its activity after pre-incubation at 50°C for just 30 min. The recombinant acetyl xylan esterase (S85-x1) showed no activity against xylan. However, xylanase activity was markedly enhanced in the presence of acetyl xylan esterase. S85-x1 can be used alongside xylanase to take advantage of this synergistic effect during industrial applications.; Xylan, a component of plant cell wall, is the most abundant non-cellulosic polysaccharide (hemicellulose) in nature. Xylanases can be use to digest xylan that has numerous use for several industries such as bioethanol production, bio-bleaching of pulp, textile, production of animal feed. In this study, we isolated many xylan-degrading microbes that were previously uncharacterized. The xylanase and acetyl xylan esterase were cloned and overexpressed in E. coli expression system, then the biochemical properties of purified recombinant enzymes were analyzed. Congo red staining method display clearing zone around xylanase positive colonies on agar plate, and this method is commonly used for investigating xylan-hydrolyzing activity. However, in this study, we adjusted microwell plate based on 3,5-dinitrosalicylic acid (DNS) method for screening xylan-degrading microbe. Decomposed xylan-containing plant and seaweed, and plant- or seaweed-consuming marine animal's intestines were sampled and screened for xylan-degrading microbes. Initial culture media were made with xylan as the sole carbon source with ground water or seawater. Subsequently isolated colonies were sub-cultured in broth containing yeast extract and xylan. Forty-four microbes that exhibit xylan hydrolyzing activity were determined by using our modified DNS method. These microbes were genetically identified, and 29 species were first reported as xylan-degrading microbes. Two strains (O. pacifica strain S85 and S. padanus strain J103) were selected for genome analysis from this pool. Whole genome sequencing was performed with GS-FLX 454 titanium. The eleven xylan-degrading relative enzymes (nine and two genes from S. padanus J103 and O. pacifica S85, respectively) were predicted by NCBI Blast algorithm. Signal peptides, domains and active sites of each genes were predicted by using web based prediction server. Two xylanase genes (J103-x2, J103-x7) from S. padanus J103 and one acetyl xylan esterase gene (S85-x1) from O. pacifica S85 were cloned and overexpressed in E. coli expression system. The recombinant enzymes were purified using ion-exchange chromatography with Ni+ charged column. Biochemical properties of purified recombinant enzymes were analyzed to establish optimum conditions. Both recombinant xylanases (J103-x2 and J103-x7) showed the highest activities at 60°C and pH 4.0. Although their optimum temperature and pH were almost similar, their thermostability showed a significant difference. The J103-x2 was stable and retaining more than 90% of its activity after pre-incubation at 50°C for 120 min, whereas J103-x7 lost most of its activity after pre-incubation at 50°C for just 30 min. The recombinant acetyl xylan esterase (S85-x1) showed no activity against xylan. However, xylanase activity was markedly enhanced in the presence of acetyl xylan esterase. S85-x1 can be used alongside xylanase to take advantage of this synergistic effect during industrial applications.