한천분해효소의 재조합발현 : 기원, 활성조건, 분비신호와 게놈분석 등

국문 초록 (Abstract)

한천분해효소(agarase)는 기초과학영역, 한천유래 고기능성 올리고당의 생산, 해조류를 이용한 바이오에너지 생산 등에 사용될 수 있다. 본 연구진은 2012년에 한천의 분류, 기원, 생산 및 응용에 관하여 총설하였다. 이에 본고에서는 2012년부터의 agarase 재조합 발현에 대해 총설하고자 한다. Agarase의 재조합 발현에 사용된 유전자는 Agarivorans 속(genus) 세균, Flameovirga 속 세균, Pseudoalteromonas 속 세균, Gayadomonas 속 세균, Catenovulum 속 세균, Microbulbifer 속 세균, Cellulophaga속 세균, Saccharophagus 속 세균, Simiduia 속, Vibrio 속 세균 등의 19종의 세균들에서 유래하였다. 47개의 재조합 발현된 agarase 중에서 α-agarase는 2개였고 나머지는 모두 β-agarase였다. α-Agarase는 모두 agarotetraose (A4)를 생산하였고 β-agarase는 NA2부터 NA12까지 다양한 산물을 생산하였다. 최적온도는 25~60℃, 최적 pH는 3.0~8.5의 범위였다. 50℃ 이상의 최적 온도를 갖는 agarase는 14개로 이들은 한천을 가열한 후에 졸상태가 유지되는 온도에서도 활발한 활성을 보일 것이다. CBM (carbohydrate-binding module)의 조작 등의 인위적 돌연변이로 agarase의 열안정성 증가, 최적온도와 활성의 동시 증가에 관한 연구사례도 있었다. 재조합발현의 숙주로 E. coli, B. subtilis, S. lividans, S. cerevisiae 등이 활용되었으며, agarase 유전자의 분비신호, 다른 생물의 분비신호 및 riboswitch가 agarase의 재조합 발현에 사용되었다. Agarase를 정제한 후에 아미노산 서열에 기반한 유전자 재조합 이외에도 게놈서열 파악과 유사성 비교를 통해 putative agarase와 메타게놈에서 유래한 agarase의 재조합 발현에 관한 연구도 있다. 이러한 연구들은 향후 agarase 및 agarase를 이용한 한천분해산물의 응용 분야 등에 활발하게 이용될 것으로 기대된다.

다국어 초록 (Multilingual Abstract)

Agarase can be used in the field of basic science, as well as for production of agar-derived high-functional oligosaccharides and bioenergy production using algae. In 2012, we summarized the classification, origin, production, and applications of agar. In this paper, we briefly review the literature on the recombinant expression of agarases from 2012 to the present. Agarase genes originated from 19 genera, including Agarivorans, Flammeovirga, Pseudoalteromonas, Gayadomonas, Catenovulum, Microbulbifer, Cellulophaga, Saccharophagus, Simiduia, and Vibrio. Of the 47 recombinant agarases, there were only two α-agarases, while the rest were β-agarases. All α-agarases produced agarotetraose, while β-agarases yielded many neoagarooligosaccharides ranging from neoagarobiose to neoagarododecaose. The optimum temperature ranged between 25 and 60℃, and the optimum pH ranged from 3.0 to 8.5. There were 14 agarases with an optimum temperature of 50℃ or higher, where agar is in sol state after melting. Artificial mutations, including manipulation of carbohydrate-binding modules (CBM), increased thermostability and simultaneously raised the optimum temperature and activity. Many hosts and secretion signals or riboswitches have been used for recombinant expression. In addition to gene recombination based on the amino acid sequence after agarase purification, recombinant expression of the putative agarase genes after genome sequencing and metagenome-derived agarases have been studied. This study is expected to be actively used in the application fields of agarase and agarase itself.

목차 (Table of Contents)

• 서론
• 결론
• References
• 초록

참고문헌 (Reference)

1 민경철, "한천분해세균 Agarivorans sp. KC-1의 분리 및 내열성 β-아가라제의 특성 규명" 한국생명과학회 28 (28): 1056-1061, 2018

2 "https://www.nongsaro.go.kr/portal/ps/psq/psqb/farm TermDicLst. ps?menu Id=PS00064"

3 "https://www.miryangagaragar.com/shop/Agar/agar05. php"

4 "http://www.agargel.com.br/agar-tec-en.html"

5 Cantarel, B. L., "The Carbohydrate-Active EnZymes database(CAZy) : an expert resource for Glycogenomics" 37 : D233-, 2009

6 Rudolph, M. M., "Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor" 159 : 1416-1422, 2013

7 Zhang, P., "Structure-based design of agarase AgWH50C from Agarivorans gilvus WH0801 to enhance thermostability" 103 : 1289-1298, 2019

8 Yoon, S. Y., "Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli" 47 : 1037-1042, 2017

9 Li, J., "Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain" 46 : 683-690, 2015

10 이지수, "Production of Ethanol from Agarose by Unified Enzymatic Saccharification and Fermentation in Recombinant Yeast" 한국미생물·생명공학회 29 (29): 625-632, 2019

11 Ramos, K. R., "Overexpression and secretion of AgaA7 from Pseudoalteromonas hodoensis sp. nov in Bacillus subtilis for the depolymerization of agarose" 90 : 19-25, 2016

12 Cui, F., "Overexpression and characterization of a novel thermostable β-agarase YM01-3, from marine bacterium Catenovulum agarivorans YM01(T)" 12 : 2731-2747, 2014

13 Lee, D. G., "Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product" 30 : 911-918, 2008

14 Su, B. M., "Mutagenesis on the surface of a β-agarase from Vibrio sp. ZC-1 increased its thermo-stability" 127 : 22-31, 2019

15 Chen, Z. W., "Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4from Paenibacillus agarexedens" 8 : 49-, 2018

16 이영득, "Molecular Cloning, Overexpression, and Enzymatic Characterization of Glycosyl Hydrolase Family 16 β-Agarase from Marine Bacterium Saccharophagus sp. AG21 in Escherichia coli" 한국미생물·생명공학회 23 (23): 913-922, 2013

17 Valverde, J. R., "Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans" 18 : 59-, 2018

18 Qu, W., "Metagenomics investigation of agarlytic genes and genomes in mangrove sediments in China, a potential repertory for carbohydrate-active enzymes" 9 : 1864-, 2018

19 Mai, Z., "Isolation and characterization of a glycosyl hydrolase family 16 β-agarase from a mangrove soil metagenomic library" 17 : E1360-, 2016

20 Yan Liu, "Isolation and Characterization of an Eosinophilic GH 16 β-Agarase (AgaDL6) from an Agar-Degrading Marine Bacterium Flammeovirga sp. HQM9" 한국미생물·생명공학회 29 (29): 235-243, 2019

21 Jeong, D. W., "Integration of bacterial expansin on agarolytic complexes to enhance the degrading activity of red algae by control of gelling properties" 20 : 1-9, 2018

22 Chen, Y. P., "Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring" 127 : 403-410, 2019

23 Seungwoo Lee, "Improvement in the Catalytic Activity of β-Agarase AgaA from Zobellia galactanivorans by Site-Directed Mutagenesis" 한국미생물·생명공학회 21 (21): 1116-1122, 2011

24 Ramos, K. R. M., "Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C" 40 : 261-267, 2018

25 Li, J., "Identification and biochemical characterization of a novel exo-type β-agarase Aga3463 from an Antarctic Pseudoalteromonas sp. strain" 129 : 162-170, 2019

26 Li, G., "Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH" 99 : 10019-10029, 2015

27 Temuujin, U., "Identification and biochemical characterization of Sco3487from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose" 194 : 142-149, 2012

28 Uyangaa Temuujin, "Identification and Characterization of a Novel β-Galactosidase from Victivallis vadensis ATCC BAA-548, an Anaerobic Fecal Bacterium" 한국미생물학회 50 (50): 1034-1040, 2012

29 Liu, H., "High expression of agarase AgaD in Escherichia coli" 55 : 1171-1176, 2015

30 Cui, X., "Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase" 120 : 657-664, 2018

31 Shi, X., "Genome sequence of the thermostable-agarase-producing marine bacterium Catenovulum agarivorans YM01(T), which reveals the presence of a series of agarase-encoding genes" 194 : 5484-, 2012

32 Kwak, M. J., "Genome sequence of the agar-degrading marine bacterium Alteromonadaceae sp. strain G7" 194 : 6961-6962, 2012

33 Dong, Q., "Genome sequence of a high agarase-producing strain Flammeovirga sp. SJP92" 12 : 13-, 2017

34 Imran, M., "Genome sequence of Microbulbifer mangrovi DD-13T reveals its versatility to degrade multiple polysaccharides" 19 : 116-124, 2017

35 Xie, Z., "Genome sequence of Cellvibrio pealriver PR1, a xylanolytic and agarolytic bacterium isolated from freshwater" 214 : 57-58, 2015

36 Lin, B., "Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105" 28 : 1691-1697, 2012

37 Liu, N., "Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801" 30 : 1691-1698, 2014

38 Alkotaini, B., "Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar" 101 : 1573-1580, 2017

39 Ko, H. J., "Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL" 78 : 3051-3058, 2012

40 Hsu, P. H., "Extracellular production of a novel endo-β-agarase AgaA from Pseudomonas vesicularis MA103 that cleaves agarose into neoagarotetraose and neoagarohexaose" 16 : 5590-5603, 2015

41 Li, L., "Extracellular expression of agarase rAgaM1 in Bacillus subtilis and its ability for neoagaro-oligosaccharide production" 59 : 359-367, 2019

42 Su, Q., "Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber" 7 : 220-, 2017

43 Liu, X., "Expression and characterization of the agarase gene aga3311 from an Antarctic bacterium" 56 : 1468-1476, 2016

44 Chen, X. L., "Expression and characterization of a novel thermostable and pH-stable β-agarase from deep-sea Bacterium Flammeovirga sp. OC4" 64 : 7251-7258, 2016

45 Gullón, S., "Exploring the feasibility of the sec route to secrete proteins using the tat route in Streptomyces lividans" 57 : 931-938, 2015

46 Alkotaini, B., "Enhanced catalytic efficiency of endo-β-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis" 93-94 : 142-149, 2016

47 Lee, M. H., "Draft genome sequence of the agarolytic haloarchaeon Halobellus rufus type strain CBA 1103" 362 : 1-3, 2015

48 Lee, M. H., "Draft genome sequence of Halolamina rubra CBA1107(T), an agarolytic haloarchaeon isolated from solar salt" 18 (18): 127-128, 2014

49 Lee, Y., "Draft genome of agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5" 21 : 13-14, 2015

50 Seok, J. H., "Construction of an expression system for the secretory production of recombinant α-agarase in yeast" 34 : 1041-1049, 2012

51 Rudolph, M. M., "Conditional control of gene expression by synthetic riboswitches in Streptomyces coelicolor" 550 : 283-299, 2015

52 정재준, "Complete genome sequence of Tamlana sp. UJ94 degrading alginate" 한국미생물학회 54 (54): 463-464, 2018

53 오지성, "Complete genome sequence of Pseudoalteromonas donghaensis HJ51T isolated from seawater" 한국미생물학회 54 (54): 305-307, 2018

54 정재준, "Complete genome sequence of Microbulbifer agarilyticus GP101 possessing genes coding for diverse polysaccharide-degrading enzymes" 한국미생물학회 54 (54): 299-301, 2018

55 Zhang, P., "Complete genome sequence of Agarivorans gilvus WH0801(T), an agarase-producing bacterium isolated from seaweed" 219 : 22-23, 2016

56 Lee, Y. S., "Complete genome sequence and analysis of three kinds of β-agarase of Cellulophaga lytica DAU203 isolated from marine sediment" 35 : 43-46, 2017

57 Xie, Z., "Comparative phenotype and genome analysis of Cellvibrio sp. PR1, a xylanolytic and agarolytic bacterium from the Pearl river" 2017 : 2017

58 Di, W., "Cloning, expression, and characterization of thermal-stable and pH-stable agarase from mangrove sediments" 58 : 302-309, 2018

59 Jung, S., "Cloning, expression, and biochemical characterization of a novel acidic GH16 β-agarase, AgaJ11, from Gayadomonas joobiniege G7" 181 : 961-971, 2017

60 Chi, W. J., "Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1" 98 : 4545-4555, 2014

61 Park, D. Y., "Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7" 175 : 733-747, 2015

62 김재덕, "Cloning, Expression, and Characterization of a Thermotolerant β-agarase from Simiduia sp. SH-4" 한국생물공학회 23 (23): 525-531, 2018

63 이동근, "Cloning, Expression, and Characterization of a Glycoside Hydrolase Family 118 β-Agarase from Agarivorans sp. JA-1" 한국미생물·생명공학회 22 (22): 1692-1697, 2012

64 Ariga, Osamu, "Cloning of Agarase Gene from Non-Marine Agarolytic Bacterium Cellvibrio sp." 한국미생물·생명공학회 22 (22): 1237-1244, 2012

65 Tawara, M., "Cloning and characterization of a novel agarase from a newly isolated bacterium Simiduia sp. strain TM-2 able to degrade various seaweeds" 177 : 610-623, 2015

66 Liu, N., "Cloning and characterisation of a novel neoagarotetraose-forming-β-agarase, AgWH50A from Agarivorans gilvus WH0801" 388 : 147-151, 2014

67 Jiao, G., "Chemical structures and bioactivities of sulfated polysaccharides from marine algae" 9 : 196-223, 2011

68 Zhang, W., "Characterization of an α-agarase from Thalassomonas sp. LD5and its hydrolysate" 102 : 2203-2212, 2018

69 Xie, W., "Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3" 97 : 4907-4915, 2013

70 Chen, X., "Characterization of a novel alkaline β-agarase and its hydrolysates of agar" 295 : 311-319, 2019

71 Choi, U., "Characterization of a Novel Neoagarobiose-producing GH42 β-Agarase, AgaJ10, from Gayadomonas joobiniege G7" 189 : 1-12, 2019

72 Liang, S. S., "Characterization and overexpression of a novel β-agarase from Thalassomonas agarivorans" 116 : 563-572, 2014

73 Wang, Y., "Characterization and overexpression of a glycosyl hydrolase family 16 β-agarase Aga0917 from Pseudoalteromonas fuliginea YTW1-15-1" 64 : 276-283, 2019

74 An, K., "Characterization and overexpression of a glycosyl hydrolase family 16 beta-agarase YM01-1 from marine bacterium Catenovulum agarivorans YM01T" 143 : 1-8, 2018

75 Chi, W. J., "Biochemical characterization of a novel iron-dependent GH16 β-agarase, AgaH92, from an agarolytic bacterium Pseudoalteromonas sp. H9" 362 : fnv035-, 2015

76 Jung, S., "Biochemical characterization of a novel cold-adapted GH39 β-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7" 101 : 1965-1974, 2017

77 Han, Z., "Biochemical characterization of a new β-agarase from Cellulophaga algicola" 20 : 2143-, 2019

78 Han, W., "Biochemical characteristics and substrate degradation pattern of a novel exo-type β-agarase from the polysaccharide-degrading marine bacterium Flammeovirga sp. strain MY04" 82 : 4944-4954, 2016

79 이영림, "Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7" 한국미생물·생명공학회 28 (28): 284-292, 2018

80 Han, W. J., "An extra peptide within the catalytic module of a β-agarase affects the agarose degradation pattern" 288 : 9519-9531, 2013

81 Lin, B., "An agarase of glycoside hydrolase family 16 from marine bacterium Aquimarina agarilytica ZC1" 364 : fnx012-, 2017

82 Liu, G., "Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities" 6 : 18726-, 2016

83 Furusawa, G., "Agarolytic bacterium Persicobacter sp. CCB-QB2 exhibited a diauxic growth involving galactose utilization pathway" 6 : e00405-, 2016

84 전명제, "Agarivorans sp. JA-1 유래 신규 GH-16 β-agarase의 클로닝, 발현 및 특성" 한국생명과학회 22 (22): 1545-1551, 2012

85 이동근, "Agarase의 분류, 기원, 확보, 활성파악, 분리정제, 생산 및 응용" 한국생명과학회 22 (22): 266-280, 2012

86 허다혜, "Agarase를 생산하는 Persicobacter sp. DH-3의 분리 및 β-agarase의 특성" 한국생명과학회 29 (29): 158-163, 2019

87 Fu, X. T., "Agarase : review of major sources, categories, purification method, enzyme characteristics and applications" 8 : 200-218, 2010

88 Dong, Q., "A β-agarase with high pH stability from Flammeovirga sp. SJP92" 432 : 1-8, 2016

89 Li, R. K., "A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris" 119 : 1164-1170, 2018

90 이영득, "A Novel Glycosyl Hydrolase Family 16 β-Agarase from the Agar- Utilizing Marine Bacterium Gilvimarinus agarilyticus JEA5: the First Molecular and Biochemical Characterization of Agarase in Genus Gilvimarinus" 한국미생물·생명공학회 28 (28): 776-783, 2018