KR101997047B1 - A beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use thereof - Google Patents

A beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use thereof Download PDF

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KR101997047B1
KR101997047B1 KR1020170160921A KR20170160921A KR101997047B1 KR 101997047 B1 KR101997047 B1 KR 101997047B1 KR 1020170160921 A KR1020170160921 A KR 1020170160921A KR 20170160921 A KR20170160921 A KR 20170160921A KR 101997047 B1 KR101997047 B1 KR 101997047B1
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이창로
홍순광
지원재
배창환
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명지대학교 산학협력단
대한민국(환경부 국립생물자원관장)
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Abstract

본 발명은 가야도모나스 주비니에게 G7 유래 베타-아가레이즈 AgaJ5 및 이의 이용에 관한 것으로, 구체적으로 가야도모나스 주비니에게 G7으로부터 동정되어 이종균주에서 과발현이 가능하며 아가로오스를 분해하여 네오아가로올리고당, 특히 네오아가로헥사오스를 주산물로 생성할 수 있고 낮은 pH 및 낮은 온도에서도 효소활성을 안정적으로 나타내는 베타-아가레이즈 AgaJ5 및 이를 이용하는 방법에 관한 것이다.
본 발명의 베타-아가레이즈는 한천을 분해하여 산업적으로 유용한 네오아가로올리고당을 생산할 수 있다. 특히 본 발명의 베타-아가레이즈는 기존의 일반적인 베타-아가레이즈와 달리 네오아가로헥사오스를 최종 분해산물로 생성하기 때문에, 우수한 생리활성을 나타내는 이 네오아가로헥사오스를 생산하는데 매우 유용하며, 특이적으로 낮은 pH 조건과 낮은 온도 조건하에서도 우수한 효소활성을 나타내고 대량생산 또한 용이하기 때문에 산업적으로 매우 유용하다.The present invention relates to a G7-derived beta-agarase AgaJ5 and its use for Gayadomonas juvinis, and more particularly to a gene for Gaya from Monacus juvinis, which is capable of overexpression in G. lucidum and decomposing agarose, Agarase AgaJ5, which is capable of producing oligosaccharides, particularly neoagarohexaose, as a main product and stably exhibiting enzyme activity even at low pH and low temperature, and a method of using the same.
The beta-agarase of the present invention can decompose agar to produce industrially useful neoagarooligosaccharides. In particular, since the beta-agarase of the present invention produces neoagarose hexaose as a final degradation product unlike the conventional beta-agarase, it is very useful for producing neoagarose hexaose exhibiting excellent physiological activity, It is industrially useful because it exhibits excellent enzyme activity even under low pH and low temperature conditions and is easy to mass-produce.

Description

가야도모나스 주비니에게 G7 유래 베타-아가레이즈 AgaJ5 및 이의 이용{A beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use thereof}Aya beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use thereof.

본 발명은 가야도모나스 주비니에게 G7 유래 베타-아가레이즈 AgaJ5 및 이의 이용에 관한 것으로, 구체적으로 가야도모나스 주비니에게 G7으로부터 동정되어 이종균주에서 과발현이 가능하며 아가로오스를 분해하여 네오아가로올리고당, 특히 네오아가로헥사오스를 주산물로 생성할 수 있고 낮은 pH 및 낮은 온도에서도 효소활성을 안정적으로 나타내는 베타-아가레이즈 AgaJ5 및 이를 이용하는 방법에 관한 것이다.The present invention relates to a G7-derived beta-agarase AgaJ5 and its use for Gayadomonas juvinis, and more particularly to a gene for Gaya from Monacus juvinis, which is capable of overexpression in G. lucidum and decomposing agarose, Agarase AgaJ5, which is capable of producing oligosaccharides, particularly neoagarohexaose, as a main product and stably exhibiting enzyme activity even at low pH and low temperature, and a method of using the same.

한천(agar)은 일부 홍조류(red algae)의 세포벽에 존재하는 다당류로, 3,6-anhydro-L-galatose 및 D-galactose로 구성되어, β-1,4 및 α-1,3-글리코시드 결합으로 번갈아 연결된 선형 사슬을 형성한다. 한천은 각각 두 가지 타입으로 분류되는 아가레이즈(agarase), 즉 베타-아가레이즈(β-agarase)와 알파-아가레이즈(α-agarase)에 의해 분해될 수 있다. 베타-아가레이즈는 한천의 β-1,4-글리코시드 결합을 특이적으로 절단하는 반면, 알파-아가레이즈는 α-1,3-글리코시드 결합만을 가수분해한다. 알파-아가레이즈가 한천을 분해하는 경우, 환원 말단에 3,6-anhydro-α-L-galactose를 갖는 아가로올리고당(agarooligosaccharide)을 생산한다. 반면, 베타-아가레이즈는 한천을 환원 말단에 D-galactose를 갖는 네오아가로올라고당(neoagarooligosaccharide)으로 분해한다. 네오아가로올리고당의 주요 성분은 두 개의 당으로 구성된 네오아가로바이오스(neoagarobiose, NA2), 네 개의 당으로 구성된 네오아가로테트라오스(neoagarotetraose, NA4) 및 여섯 개의 당으로 구성된 네오아가로헥사오스(neoagarohexaose, NA6)이다. NA2는 네오아가로바이오스 가수분해효소(neoagarobiose hydrolase, NABH)에 의해 단량체 당인 3,6-anhydro-L-galatose와 D-galactose로 분해될 수 있다. 두 단량체 당 중에서 D-galactose는 박테리아 세포의 탄소 공급원으로 더 쉽게 사용될 수 있다. 다른 당인 3,6-anhydro-L-galatose를 탄소원으로 활용하기 위해서는 추가적인 효소분해를 통해 2-keto-3-deoxy-galactonate로의 전환이 필요하다. 이것은 두 개의 촉매효소, 즉 NADP+-의존형 3,6-anhydro-α-L-galactose dehydrogenase와 3,6-anhydrogalactonate cycloisomerase의 순차적인 작용에 의해 이루어진다. 한천의 분해와 관련된 이화 효소에 대한 이해는 한천을 산업적인 화학물질 또는 생물연료로 생물전환시키는 기술을 개발하는데 필요하다. 특히, 아가레이즈가 생산하는 네오아가로올리고당은 항균 활성, 피부 보습, 흑색종 세포의 미백, 항비만 활성, 항당뇨병 활성과 같은 많은 생물학적 활성을 갖는 것으로 알려져 있다.Agar is a polysaccharide present in the cell wall of some red algae and consists of 3,6-anhydro-L-galactose and D-galactose, and is composed of β-1,4 and α-1,3-glycosides Bond to form alternating linear chains. Agar can be degraded by two types of agarases, beta-agarase and alpha-agarase, respectively. The beta-agarase specifically cleaves the? -1,4-glycosidic linkage of agar while the alpha-agarase hydrolyzes only the? -1,3-glycosidic linkage. When alpha-agarase degrades agar, it produces agarooligosaccharide with 3,6-anhydro-α-L-galactose at the reducing end. Beta-agarase, on the other hand, degrades agar into neoagarooligosaccharides by neoagarose with D-galactose at the reducing end. The main component of neoagarooligosaccharide is neoagarobiose (NA2) composed of two sugars, neoagarotetraose (NA4) composed of four sugars and neoagarohexaose composed of six sugars neoagarohexaose, NA6). NA2 can be cleaved by the neoagarobiose hydrolase (NABH) into 3,6-anhydro-L-galactose and D-galactose, which are monomers. Among the two monomeric sugars, D-galactose can be used more easily as a carbon source for bacterial cells. In order to utilize the other sugar, 3,6-anhydro-L-galatose as a carbon source, the conversion to 2-keto-3-deoxy-galactonate is necessary through additional enzymatic digestion. This is accomplished by the sequential action of two catalytic enzymes, NADP + -dependent 3,6-anhydro-α-L-galactose dehydrogenase and 3,6-anhydrogalactonate cycloisomerase. Understanding the lysing enzymes involved in the degradation of agar is needed to develop techniques for bioconversion of agar into industrial chemicals or biofuels. In particular, neoagarooligosaccharides produced by Agarase are known to have many biological activities such as antibacterial activity, skin moisturization, whitening of melanoma cells, anti-obesity activity, and anti-diabetic activity.

베타-아가레이즈는 Alteromonas, Pseudomonas, Gayadomonas, Vibrio, Pseudoalteromonas, Zobellia, Catenovulum, Agarivorans, Cohnella, Bacillus, Thalassomonas, Streptomyces, Stenotrophomonas, Simiduia, Flammeovirga 등의 다양한 세균에서 발견되었으며, 각각 GH16, GH39, GH50, GH86 및 GH118을 포함하는 여러 glycoside hydrolase(GH) 패밀리로 분류되었다. 다양한 GH16 베타-아가레이즈가 생화학적 분석을 통해 광범위하게 연구되었으나, GH86과 같은 다른 GH 패밀리에서는 소수의 베타-아가레이즈 만이 생화학적으로 특성화되어 있어, 이들 패밀리에 속하는 새로운 베타-아가레이즈의 발굴 및 생화학적 특성에 관한 연구가 필요하다. 또한, 기존에 알려진 베타-아가레이즈는 대부분 최종 분해산물이 NA2이므로 NA4 또는 NA6를 생산하기 위해서는 완전한 분해가 이루어지지 않도록 효소활성을 조절해야 하는 등 비효율적이라는 문제가 있다. 따라서 이러한 문제를 해소할 수 있는 새로운 베타-아가레이즈, 즉 NA4 또는 NA6를 최종분해산물로 생성할 수 있는 효소의 발굴이 필요하다.Beta-agarase is Alteromonas, Pseudomonas, Gayadomonas, Vibrio, Pseudoalteromonas, Zobellia, Catenovulum, Agarivorans, Cohnella, Bacillus, Thalassomonas, Streptomyces, Stenotrophomonas, Simiduia, were found in a variety of bacteria, such as Flammeovirga, each of GH16, GH39, GH50, GH86 And several glycoside hydrolase (GH) families including GH118. Although various GH16 beta-agarases have been extensively studied through biochemical analysis, only a few beta-agarases have been biochemically characterized in other GH families such as GH86, and new beta-agarases belonging to these families have been discovered and Studies on biochemical properties are needed. In addition, since most of the known beta-agarase products are NA2, there is a problem in that it is inefficient to control the enzyme activity so as to prevent complete decomposition in order to produce NA4 or NA6. Therefore, it is necessary to discover an enzyme capable of producing a new beta-agarase, i.e., NA4 or NA6, as a final degradation product, which can solve this problem.

이에 본 발명자는 한천을 분해하여 산업적으로 유용하게 활용하는데 필요한 보다 효과적이고 특이적인 성질을 갖는 새로운 효소, 특히 NA4 또는 NA6를 최종분해산물로 생성할 수 있는 새로운 효소를 발굴하고자 노력하였으며, 이에 우리나라 가야섬의 연안 바닷물에서 분리된 가야도모나스 주비니에게 G7(Gayadomonas joobiniege G7) 균주를 이용하였다.Therefore, the present inventor has sought to discover a novel enzyme capable of producing a more effective and specific new enzyme, particularly NA4 or NA6, as a final degradation product, which is necessary for decomposing agar and industrially usefully. Gaya ( Gayadomonas joobiniege G7) strains were used for Ganoderma lucidum isolates from coastal waters of the island.

가야도모나스 주비니에게 G7(Gayadomonas joobiniege G7)은 가야 섬(Gaya Island)의 해수 샘플에서 분리된 막대 모양의 호기성, 비 운동성 및 비-색소성 그람 음성 해양 세균이며, 강한 한천 분해 활성을 가지고 있다. G. joobiniege G7의 16S rRNA 유전자 염기서열은 Catenovulum agarivorans YM01과 95.5%의 유사성을 나타내며, 전체 게놈 서열 분석결과 GH16, GH39, GH42 및 GH86에 속하는 12개의 아가레이즈 유전자가 있는 것으로 추정되었다. 그 중 두 가지 베타-아가레이즈가 생화학적으로 특징 지어졌다. 이중 하나인 GH39에 속하는 베타-아가레이즈 AgaJ9는 5℃에서 80% 이상의 활성을 갖는 내한성 효소이며, 다른 하나인 GH16 베타-아가레이즈 AgaJ11은 pH 4 ~ 5의 산성 조건에서만 활성을 나타내는 산성 베타-아가레이즈이다. 이들 두 가지 베타-아가레이즈 이외에 나머지 유전자들은 별도의 연구가 이루어지지 않았기 때문에 어떤 활성을 갖는 효소를 코딩하는 유전자인지 알 수 없는 실정이다. Gayadomonasubinyi G7 ( Gayadomonas joobiniege G7) is a rod-shaped aerobic, non-motile and non-pigmented Gram-negative marine bacterium isolated from seawater samples from Gaya Island and has strong agar-degrading activity . The 16S rRNA gene sequence of G. joobiniege G7 showed 95.5% similarity with Catenovulum agarivorans YM01. It was estimated that there were 12 agarase genes belonging to GH16, GH39, GH42 and GH86 as a result of whole genome sequence analysis. Two of these beta - agarases were biochemically characterized. The beta-agarase AgaJ9 belonging to GH39 belonging to one of them is a cold tolerant enzyme having an activity of 80% or more at 5 DEG C and the other GH16 beta-agarase AgaJ11 is an acid beta-agar which exhibits activity only at an acidic condition of pH 4 to 5 It raises. In addition to these two types of beta-agarase, the remaining genes have not been studied so that it is impossible to know the gene encoding an enzyme having an activity.

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따라서 본 발명의 주된 목적은 한천을 분해하여 산업적으로 유용하게 활용하는데 필요한 보다 효과적이고 특이적인 성질을 갖는 새로운 효소를 제공하는데 있다. 특히 NA2가 아닌 NA4 또는 NA6를 최종분해산물로 생성할 수 있으며, 특이적인 환경, 예를 들어 산성 또는 알칼리성 조건, 낮거나 높은 온도 조건에서 베타-아가레이즈 활성을 나타내며 대량생산이 용이한 새로운 효소를 제공하는데 있다.Therefore, it is a main object of the present invention to provide a new enzyme having more effective and specific properties necessary for decomposing agar and industrially utilizing it. In particular, NA4 or NA6, which is not NA2, can be produced as a final degradation product, and a novel enzyme that exhibits a beta-agarase activity at a specific environment, for example, acidic or alkaline conditions, .

본 발명의 다른 목적은 상기와 같은 효소를 함유하여 산업적으로 유용한 용도로 사용할 수 있는 효소조성물, 상기 효소의 생산에 필요한 유전자, 재조합 벡터, 형질전환체 및 이들을 이용한 상기 효소의 대량생산 방법과 상기 효소를 산업적으로 유용하게 이용하는 방법을 제공하는데 있다.It is another object of the present invention to provide an enzyme composition which can be used for industrially useful applications by containing such an enzyme, a gene necessary for the production of the enzyme, a recombinant vector, a transformant and a method for mass- The present invention provides a method for industrially advantageous use of the method.

본 발명의 한 양태에 따르면, 본 발명은 서열번호 1의 아미노산 서열을 갖는 베타-아가레이즈(beta-agarase)를 제공한다.According to one aspect of the invention, the invention provides a beta-agarase having the amino acid sequence of SEQ ID NO: 1.

본 발명의 다른 양태에 따르면, 본 발명은 상기 베타-아가레이즈를 유효성분으로 함유하는 네오아가로헥사오스(neoagaroehexaose) 생산용 효소조성물을 제공한다.According to another aspect of the present invention, there is provided an enzyme composition for producing neoagaroehexaose containing the above-mentioned beta-agarase as an active ingredient.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 베타-아가레이즈를 유효성분으로 함유하는 아가로오스(agarose) 분해용 효소조성물을 제공한다.According to still another aspect of the present invention, there is provided an enzyme composition for agarose degradation comprising the beta-agarase as an active ingredient.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 베타-아가레이즈를 코딩하는 베타-아가레이즈 유전자를 제공한다.According to another aspect of the present invention, there is provided a beta-agarase gene encoding said beta-agarase.

본 발명의 베타-아가레이즈 유전자는 서열번호 4의 염기서열을 갖는 것이 바람직하다.The beta-agarase gene of the present invention preferably has the nucleotide sequence of SEQ ID NO: 4.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 유전자를 함유하는 베타-아가레이즈 생산용 재조합 벡터를 제공한다.According to still another aspect of the present invention, there is provided a recombinant vector for producing a beta-agarase containing the gene.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 재조합 벡터로 형질전환된 베타-아가레이즈 생산용 형질전환체를 제공한다.According to another embodiment of the present invention, the present invention provides a transformant for producing beta-agarase transformed with said recombinant vector.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 형질전환체를 배양하고 상기 베타-아가레이즈 유전자를 과발현시키는 것을 특징으로 하는 베타-아가레이즈 대량생산방법을 제공한다.According to still another aspect of the present invention, there is provided a method for mass production of beta-agarase characterized by culturing the transformant and overexpressing the beta-agarase gene.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 베타-아가레이즈를 아가로오스와 효소반응시키는 것을 특징으로 하는 네오아가로헥사오스 생산방법을 제공한다.According to another aspect of the present invention, there is provided a method for producing neoagarohexaose, wherein the beta-agarase is reacted with agarose by an enzyme.

본 발명의 네오아가로헥사오스 생산방법에 있어서, 상기 효소반응이 pH 4 내지 6에서 이루어지는 것이 바람직하다.In the neoagarohexaose production method of the present invention, it is preferable that the enzyme reaction is carried out at a pH of 4 to 6.

본 발명의 네오아가로헥사오스 생산방법에 있어서, 상기 효소반응이 10 내지 40℃에서 이루어지는 것이 바람직하다.In the neoagarohexaose production method of the present invention, the enzyme reaction is preferably performed at 10 to 40 ° C.

본 발명의 또 다른 양태에 따르면, 본 발명은 상기 베타-아가레이즈를 아가로오스와 효소반응시키는 것을 특징으로 하는 아가로오스 분해방법을 제공한다.According to another aspect of the present invention, there is provided a method for degrading agarose characterized by reacting the beta-agarase with agarose.

본 발명의 아가로오스 분해방법에 있어서, 상기 효소반응이 pH 4 내지 6에서 이루어지는 것이 바람직하다.In the agarose degradation method of the present invention, the enzyme reaction is preferably carried out at a pH of 4 to 6. [

본 발명의 아가로오스 분해방법에 있어서, 상기 효소반응이 10 내지 40℃에서 이루어지는 것이 바람직하다.In the agarose degradation method of the present invention, the enzyme reaction is preferably performed at 10 to 40 캜.

본 발명의 베타-아가레이즈는 한천을 분해하여 산업적으로 유용한 네오아가로올리고당을 생산할 수 있다. 특히 본 발명의 베타-아가레이즈는 기존의 일반적인 베타-아가레이즈와 달리 네오아가로헥사오스를 최종 분해산물로 생성하기 때문에, 우수한 생리활성을 나타내는 이 네오아가로헥사오스를 생산하는데 매우 유용하며, 특이적으로 낮은 pH 조건과 낮은 온도 조건하에서도 우수한 효소활성을 나타내고 대량생산 또한 용이하기 때문에 산업적으로 매우 유용하다.The beta-agarase of the present invention can decompose agar to produce industrially useful neoagarooligosaccharides. In particular, since the beta-agarase of the present invention produces neoagarose hexaose as a final degradation product unlike the conventional beta-agarase, it is very useful for producing neoagarose hexaose exhibiting excellent physiological activity, It is industrially useful because it exhibits excellent enzyme activity even under low pH and low temperature conditions and is easy to mass-produce.

도 1은 본 발명의 일실시예에 따라 정제된 재조합 AgaJ5를 SDS-PAGE 겔 상에서 분리한 것(A)과 자이모그램 분석한 결과(B)이다. Lane M, size marker; lane 1, pHis-AgaJ5 재조합 벡터로 형질전환된 E. coli ER2566의 인덕션(induction)하기 전 세포 추출물; lane 2, pHis-AgaJ5 재조합 벡터로 형질전환된 E. coli ER2566을 1mM IPTG로 인덕션한 이후의 세포 추출물; lane 3, 세포 조추출물의 수용성 분획물; lane 4, 정제된 AgaJ5.
도 2는 본 발명의 일실시예에 따라 정제된 재조합 AgaJ5의 효소활성에 pH(A), 온도(B) 및 금속이온(C)이 미치는 영향을 조사한 결과이다.
도 3은 본 발명의 일실시예에 따라 정제된 재조합 AgaJ5의 동역학적 계수를 결정하기 위한 그래프이다. 모든 데이터는 적어도 2회의 반복실험을 통한 평균값으로 나타냄.
도 4는 본 발명의 일실시예에 따라 정제된 재조합 AgaJ5의 반응산물을 분석한 결과이다. (A) 시간별 반응산물의 점성도 측정 결과, (B) 시간별 아가로오스 가수분해산물의 TLC 분석 결과, (C) 다양한 네오아가로올리고당을 기질로 이용한 반응산물의 TLC 분석 결과. NA2, 네오아가로바이오스(neoagarobiose); NA4, 네오아가로테트라오스(neoagarotetraose); NA6, 네오아가로헥사오스(neoagarohexaose).
도 5 및 6은 본 발명의 일실시예에 따라 정제된 재조합 AgaJ5에 의한 가수분해산물의 MALDI-TOF 분석 결과이다. 도 5는 TLC 상 NA4 spot의 분석 결과, 도 6은 TLC 상 NA6 spot의 분석 결과.FIG. 1 is a result of separation of purified recombinant AgaJ5 on an SDS-PAGE gel according to an embodiment of the present invention and FIG. Lane M, size marker; lane 1, cell extract before induction of E. coli ER2566 transformed with the pHis-AgaJ5 recombinant vector; lane 2, cell extract after inoculation of E. coli ER2566 transformed with the pHis-AgaJ5 recombinant vector with 1 mM IPTG; lane 3, soluble fraction of cell-cell extract; lane 4, purified AgaJ5.
2 is a graph showing the effect of pH (A), temperature (B) and metal ion (C) on the enzyme activity of the purified recombinant AgaJ5 according to an embodiment of the present invention.
Figure 3 is a graph for determining the kinetic coefficients of purified recombinant AgaJ5 according to one embodiment of the present invention. All data are expressed as mean values through at least two repeated experiments.
FIG. 4 is a result of analyzing the reaction product of the purified recombinant AgaJ5 according to an embodiment of the present invention. (A) Results of TLC analysis of reaction products with time, (B) Results of TLC analysis of agarose hydrolysis products over time, (C) TLC analysis results of reaction products using various neoagarooligosaccharides as substrates. NA2, neoagarobiose; NA4, neoagarotetraose; NA6, neoagarohexaose.
Figures 5 and 6 are MALDI-TOF analysis results of the hydrolyzate by purified recombinant AgaJ5 according to one embodiment of the present invention. FIG. 5 shows an analysis result of NA4 spot on TLC, and FIG. 6 shows an analysis result of NA6 spot on TLC.

본 발명의 베타-아가레이즈 AgaJ5는 가야도모나스 주비니에게 G7(Gayadomonas joobiniege G7)의 게놈 염기서열 분석과정에서 나타난 가상의 단백질(hypothetical protein)로 지금까지 그 기능이 밝혀져지지 않았었으나, 본 발명을 통해 베타-아가레이즈임이 밝혀졌다. 이에 본 발명은 새롭게 밝혀진 AgaJ5의 특성을 이용하는 것을 특징으로 한다.The beta-agarase AgaJ5 of the present invention is a hypothetical protein revealed in the genome sequence analysis of G7 ( Gayadomonas joobiniege G7) to Gayadomonas jubini , but its function has not been revealed so far. However, Beta-agarase. Therefore, the present invention is characterized by using the newly discovered characteristic of AgaJ5.

AgaJ5의 생산균주인 가야도모나스 주비니에게 G7은 우리나라 가야섬의 연안 바닷물에서 분리된 균주로 한국생명공학연구원 생물자원센터에 KCTC23721로, 미국균주은행(American Type Culture Collection, ATCC)에 ATCC BAA-2321로, 독일균주은행(Deutsche Sammlung von Microorganismund Zellkulturen Gmb H, DSM)에 DSM25250로 기탁되어 있다.Gaya, a producer strain of AgaJ5, was isolated from coastal waters of Gaya Island in Korea and was deposited with the American Type Culture Collection (ATCC) as KCTC23721 at the Korea Research Institute of Bioscience and Biotechnology, ATCC BAA- 2321, deposited with DSM 25250 in Deutsche Sammlung von Microorganismund Zellkulturen Gmb H, DSM.

AgaJ5는 처음에는 서열번호 2의 아미노산 서열로 이루어지는 전구단백질(pre-mature protein)로 발현되고 이후 시그널 펩티드(signal peptide)(서열번호 2의 1 ~ 30번 아미노산)가 잘려 서열번호 1의 아미노산 서열로 이루어진 성숙단백질(mature protein)이 될 것으로 예상되었다. 따라서 시그널 펩티드가 제외된 서열번호 1의 아미노산 서열만으로 이루어지더라도 본래의 활성을 나타낼 수 있을 것으로 예상되었으며, 본 발명에서 실험을 통해 이를 입증하였다. 본 발명에서는 서열번호 1의 아미노산 서열의 N 말단 부위에 다수의 히스티딘(Histidine)이 연결된 형태(서열번호 3)로 제조되었지만, 이 부위는 단백질의 정제를 용이하게 하기 위한 부분으로 본래 단백질의 활성에는 영향을 미치지 않는다. 따라서 AgaJ5의 베타-아가레이즈 활성을 이용하기 위해서는 서열번호 1의 아미노산 서열만으로 이루어지더라도 가능하며, 이 밖에 N-말단 또는 C-말단에 다양한 아미노산 서열을 부가하는 것도 가능할 것이다. 부가되는 서열에 특별한 제한은 없으나 표적화 서열, 태그(tag), 표지된 잔기, 반감기 또는 펩티드의 안정성을 증가시키기 위한 특정 목적으로 고안된 아미노산 서열 등을 부가하는 것이 바람직할 것이다. 예를 들어, 정제의 용이성을 위해 다수개의 히스티딘(Histidine)을 N-말단 또는 C-말단에 부가한 형태일 수 있다(서열번호 3 참조). 또한 전구단백질과 같이 시그널 펩티드가 포함된 형태로도 이용할 수 있을 것이다.AgaJ5 is first expressed as a pre-mature protein consisting of the amino acid sequence of SEQ ID NO: 2 and then the signal peptide (amino acids 1-30 of SEQ ID NO: 2) is truncated to form the amino acid sequence of SEQ ID NO: It was expected to be a mature protein made. Therefore, it was expected that even if the amino acid sequence of SEQ ID NO: 1 was excluded from the signal peptide, it could exhibit its original activity, and this was proved through experiments in the present invention. In the present invention, a plurality of histidine is connected to the N-terminal region of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 3), but this region is a part for facilitating the purification of the protein. It does not affect. Therefore, in order to utilize the beta-agarase activity of AgaJ5, it is possible to use only the amino acid sequence of SEQ ID NO: 1, and it is also possible to add various amino acid sequences to the N-terminal or C-terminal. The sequence to be added is not particularly limited, but it may be desirable to add targeting sequences, tags, labeled residues, half-lives, or amino acid sequences designed for specific purposes to increase the stability of the peptides. For example, a number of histidine may be added to the N-terminus or C-terminus for ease of purification (see SEQ ID NO: 3). It may also be used in the form of a signal peptide, such as a progenitor protein.

본 발명에 따르면, AgaJ5는 endo-작용성 베타-아가레이즈이며, 아가로오스를 분해하여 여섯 개의 당분자가 연결되어 이루어진 네오아가로헥사오스(neoagarohexaose; NA6)를 주산물로 생성한다. 아가로오스의 분해 과정에서 소량의 네오아가로바이오스(neoagarobiose; NA2) 및 네오아가로테트라오스(neoagarotetraose; NA4)가 생성될 수 있으나, 이는 여덟 개 또는 열 개의 당분자가 연결되어 이루어진 네오아가로옥타오스(neoagarooctaose, NA8) 또는 네오아가로데카오스(neoagarodecaose, NA10)를 AgaJ5가 분해할 때 생성되는 것으로 주분해산물은 아니다. 따라서 본 발명의 AgaJ5는 네오아가로올리고당 중에서도 네오아가로헥사오스의 생산에 매우 유용하다.According to the present invention, AgaJ5 is an endo-functional beta-agarase, which produces neoagarohexaose (NA6) as a main product in which agarose is degraded and six sugar molecules are connected. A small amount of neoagarobiose (NA2) and neoagarotetraose (NA4) can be produced in the degradation process of agarose, but it is possible that neoagarotaxa It is produced when AgaJ5 breaks down neoagarooctaose (NA8) or neoagarodecaose (NA10), and it is not a perennial seafood. Therefore, AgaJ5 of the present invention is very useful for the production of neoagarohexaose among neoagarooligosaccharides.

본 발명에 따르면, AgaJ5는 pH 4 내지 6의 조건 및 10 내지 40℃의 조건에서 보다 우수한 베타-아가레이즈 활성을 나타낼 수 있다. 따라서 보다 효율적인 효소반응을 위해서는 상기와 같은 pH 및 온도 조건을 적용하는 것이 바람직하다. 보다 바람직하게는 pH 4.5 내지 5.5, 25 내지 35℃의 조건을 적용하는 것이 바람직하다.According to the present invention, AgaJ5 can exhibit better beta-agarase activity at pH 4 to 6 and at 10 to 40 ° C. Therefore, it is preferable to apply the pH and temperature conditions as described above for a more efficient enzyme reaction. More preferably, the conditions of pH 4.5 to 5.5 and 25 to 35 DEG C are applied.

상기와 같은 AgaJ5의 특성을 이용한 본 발명의 네오아가로헥사오스 생산용 효소 조성물은 AgaJ5 또는 이의 재조합 단백질 이외에도 단백질의 효소활성을 안정적으로 유지할 수 있도록 하기 위한 완충제, 또는 부형제 등의 첨가제를 포함할 수 있으며, 동일 또는 유사한 효소활성을 갖는 다른 효소도 추가로 포함될 수 있을 것이다.The enzyme composition for producing neoagaroohexaose of the present invention using the characteristics of AgaJ5 may include additives such as buffering agents or excipients to stably maintain the enzyme activity of AgaJ5 or a recombinant protein thereof And other enzymes having the same or similar enzymatic activity may be further included.

상기와 같이 AgaJ5는 고분자의 아가로오스를 상대적으로 저분자 형태인 네오아가로헥사오스로 효과적으로 분해할 수 있고, 이에 따라 아가로오스를 분해하기 위한 용도로도 유용하게 활용될 수 있다. 아가로오스를 분해하는 활성은 아가로오스를 이용한 DNA 또는 RNA와 같은 분자의 전기영동 이후 아가로오스 겔로부터 이들 분자를 회수하는데 유용하게 활용될 수 있다. 따라서 AgaJ5 또는 이의 재조합 단백질을 아가로오스 겔로부터 DNA를 추출하기 위한 용도의 효소 조성물 또는 키트에 이용할 수 있다. 예를 들어 DNA gel extraction kit 등에 이용할 수 있다. 이때 키트에는 AgaJ5 또는 이의 재조합 단백질 이외에도 DNA 추출 시 필요한 시약 또는 기구 등이 추가로 포함될 수 있을 것이다.As described above, AgaJ5 can effectively decompose agarose of a polymer into neoagarose hexaose, which is a relatively low molecular form, and thus can be usefully used for decomposing agarose. The activity of degrading agarose may be useful for recovering these molecules from agarose gel after electrophoresis of molecules such as DNA or RNA using agarose. Therefore, AgaJ5 or a recombinant protein thereof can be used as an enzyme composition or kit for extracting DNA from an agarose gel. For example, DNA gel extraction kit can be used. In addition to AgaJ5 or a recombinant protein thereof, the kit may further include reagents or instruments necessary for DNA extraction.

가야도모나스 주비니에게 G7의 게놈 상에는 전구단백질 형태의 AgaJ5가 서열번호 5의 염기서열로 암호화되어 있다. 하지만 위에서 설명한 바와 같이 베타-아가레이즈 활성은 시그널 펩티드가 잘린 형태에서도 발휘되므로, 서열번호 5의 염기서열에서 시그널 펩티드를 암호화하는 부위가 제거된 서열번호 4의 염기서열이 발현되더라도 발현된 단백질은 베타-아가레이즈 활성을 나타낼 수 있다. 또한 반드시 서열번호 4와 같은 염기서열이 아니더라도 같은 아미노산 서열을 암호화하도록 변경된 서열이 발현되면 발현된 단백질은 베타-아가레이즈 활성을 나타낼 수 있다. 예를 들어, 서열번호 4에서 코돈 서열을 변형하는 방법이 여기에 해당될 수 있다. 즉, 서열번호 1의 아미노산 서열을 암호화하는 유전자가 발현되면 발현된 단백질은 베타-아가레이즈 활성을 나타낼 수 있을 것이다.To Gaya monosuvenia, AgaJ5 in the form of a precursor protein is encoded in the nucleotide sequence of SEQ ID NO: 5 in the genome of G7. However, as described above, since the beta-agarase activity is exerted even in the form of the cleaved signal peptide, even if the nucleotide sequence of SEQ ID NO: 5, in which the region coding for the signal peptide is removed, is expressed, - can indicate agarase activity. In addition, if the sequence is changed so as to encode the same amino acid sequence even if it is not the nucleotide sequence shown in SEQ ID NO: 4, the expressed protein may exhibit the beta-agarase activity. For example, this may be the method of modifying the codon sequence in SEQ ID NO: 4. That is, when the gene encoding the amino acid sequence of SEQ ID NO: 1 is expressed, the expressed protein may exhibit beta-agarase activity.

본 발명의 재조합 벡터는 상기와 같은 서열번호 1의 아미노산 서열을 암호화하는 유전자를 함유함으로써 베타-아가레이즈를 생산하기 위해 이용될 수 있는데, 이때 이 유전자에는 염기서열의 5'-말단 또는 3'-말단에 다양한 염기서열이 부가될 수 있다. 부가되는 서열로는 위에서 언급한 서열번호 1의 아미노산 서열의 N-말단 또는 C-말단에 부가될 수 있는 아미노산 서열을 암호화하는 염기서열일 수 있으며, 여기에는 표적화 서열, 태그(tag), 표지된 잔기, 반감기 또는 펩티드의 안정성을 증가시키기 위한 특정 목적으로 고안된 아미노산 서열 등을 암호화하는 서열이 포함된다. 예를 들어, 정제의 용이성을 위해 다수개의 히스티딘(Histidine)을 암호화하는 서열을 5'-말단 또는 3'-말단에 부가한 형태일 수 있다(서열번호 6 참조). 또한 상기 유전자로는 본래 AgaJ5를 암호화하는 유전자(서열번호 5 참조)를 이용할 수도 있다.The recombinant vector of the present invention can be used to produce a beta-agarase by containing a gene encoding the amino acid sequence of SEQ ID NO: 1, wherein the gene has 5'-terminal or 3'- Various base sequences may be added to the ends. The sequence to be added may be a nucleotide sequence encoding an amino acid sequence that can be added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1 mentioned above, and includes a targeting sequence, a tag, Residues, half-life, or amino acid sequences designed for specific purposes to increase the stability of the peptide, and the like. For example, a sequence encoding a plurality of histidine may be added at the 5'-end or 3'-end for ease of purification (see SEQ ID NO: 6). As the above-mentioned gene, a gene encoding AgaJ5 (see SEQ ID NO: 5) may be used.

본 발명의 재조합 벡터는 상기와 같은 유전자가 용이하게 발현될 수 있도록 숙주에 따라 적합한 프로모터(promoter) 및 터미네이터(terminator)가 포함되는 것이 바람직하며, 이 프로모터 및 터미네이터로는 agaJ5 고유의 프로모터 및 터미네이터를 이용할 수도 있다. 이러한 프로모터 및 터미네이터가 포함되어 있으며 외래 유전자의 도입이 용이하게 설계된 다양한 벡터들이 개발되어 있어 이들을 이용할 수 있다. 예를 들어 대장균(Escherichia coli)을 숙주로 이용할 경우 pET 시리즈의 벡터를 이용할 수 있다.The recombinant vector of the present invention preferably includes a suitable promoter and a terminator depending on the host so that the gene can be easily expressed. The promoter and terminator include a promoter and a terminator unique to agaJ5 , It can also be used. Various vectors containing such promoters and terminators have been developed and are easily designed for the introduction of foreign genes, so that they can be used. For example, when Escherichia coli is used as a host, vectors of the pET series can be used.

본 발명의 형질전환체는 상기와 같은 재조합 벡터를 숙주생물체에 도입하여 제조될 수 있다. 이때 숙주로는 다양한 생물체를 이용할 수 있을 것으로 판단되며, 베타-아가레이즈의 안정적인 발현 또는 생산 효율을 위해 미생물을 이용하는 것이 보다 바람직할 것이다. 본 발명에 따르면 agaJ5가 대장균의 발현시스템에서도 매우 원활하게 발현될 수 있는 것으로 확인되었다. 따라서 대장균을 숙주로 이용하여 형질전환체를 제조하면 베타-아가레이즈을 매우 용이하게 생산할 수 있다. 숙주생물체의 형질전환은 각 숙주생물체에 이용되고 있는 통상의 형질전환방법을 적용하여 상기 재조합 벡터를 숙주생물체에 도입하는 방법으로 달성할 수 있다.The transformant of the present invention can be produced by introducing such a recombinant vector into a host organism. At this time, it is considered that various hosts can be used as the host, and it is more preferable to use microorganisms for stable expression or production efficiency of the beta-agarase. It has been confirmed according to the present invention that agaJ5 can be expressed very smoothly also in the expression system of E. coli. Therefore, when a transformant is prepared using E. coli as a host, beta-agarase can be produced very easily. Transformation of the host organism can be accomplished by introducing the recombinant vector into the host organism by applying conventional transformation methods used in each host organism.

본 발명의 베타-아가레이즈 대량생산은 상기 형질전환체를 배양하고 베타-아가레이즈 유전자를 과발현시키는 방법으로 달성할 수 있다. 이때 배양배지의 종류, 배양온도, 배양시간 등의 조건은 숙주 및 벡터의 종류에 따라 선택적으로 적용할 수 있다. 예를 들어 숙주가 대장균이고 pET28 시리즈의 벡터를 이용하는 경우, LB(Luria Bertani) 배지에서 약 15 ~ 40℃로 12시간 ~ 5일간 배양하는 방법을 사용할 수 있다.The mass production of the beta-agarase of the present invention can be achieved by culturing the transformant and overexpressing the beta-agarase gene. At this time, conditions such as the type of culture medium, culture temperature, and incubation time can be selectively applied depending on the host and vector. For example, when the host is Escherichia coli and the vector of pET28 series is used, a method of culturing in LB (Luria Bertani) medium at about 15 to 40 DEG C for 12 hours to 5 days can be used.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[실시예] 베타-아가레이즈 AgaJ5 생산 및 특성분석EXAMPLES Production and characterization of beta-agarase AgaJ5

1. 방법1. Method

1-1. 균주 및 배양조건1-1. Strain and culture conditions

Escherichia coli strain ER2566을 AgaJ5의 클로닝 및 발현에 사용하고 Luria-Bertani(LB) 배지에서 37℃에서 성장시켰다. pET-28a 플라스미드로 형질전환한 ER2566의 경우, 카나마이신(50㎍/㎖)을 첨가하였다. Escherichia coli strain ER2566 was used for cloning and expression of AgaJ5 and grown at 37 ° C in Luria-Bertani (LB) medium. For ER2566 transformed with the pET-28a plasmid, kanamycin (50 占 퐂 / ml) was added.

1-2. 유전자 클로닝1-2. Gene cloning

pHis-AgaJ5(히스티딘이 태깅된 형태의 AgaJ5를 생산하기 위한 재조합 벡터)를 제조하기 위해, AgaJ5(NCBI 참조 서열 : WP_017446675.1)에서 예상 시그널 펩티드(서열번호 1의 아미노산 서열에서 1 ~ 30번 아미노산)가 제외된 형태(서열번호 1)의 유전자 단편(서열번호 4)을 pET-28a 플라스미드에 클로닝 하였다. 이 2,328bp의 유전자 단편을 증폭하기 위해, 정방향 프라이머(5'-CGCGCGGCAGCCATATGACTAAAGAACCAACTGCCGT-3') 및 역방향 프라이머(5'-GCTCGAATTCGGATCCAGTAAAGGAGTGAGTGCAAA-3')를 사용하여 중합효소연쇄반응(PCR)을 수행하였다. 이들 프라이머는 In-Fusion 반응을 위한 NdeI 및 BamHI 제한효소로 절단된 pET28a 절편의 말단 15bp 서열에 상응하는 어댑터 서열(밑줄)을 함유한다. PCR은 Q-Cycler(Quanta Biotech, England)를 사용하여 수행하였다. PCR 증폭물을 In-Fusion HD 클로닝 키트(Clontech, USA)를 사용하여 NdeI 및 BamHI 제한효소로 절단된 pET28a 절편에 삽입하였다.In order to prepare pHis-AgaJ5 (a recombinant vector for producing histidine-tagged AgaJ5), the predicted signal peptide (the amino acid sequence from amino acid 1 to 30 in the amino acid sequence of SEQ ID NO: 1) in AgaJ5 (NCBI reference sequence: WP017446675.1) ) (SEQ ID NO: 1) (SEQ ID NO: 4) was cloned into the pET-28a plasmid. Polymerase chain reaction (PCR) was performed using a forward primer (5'- CGCGCGGCAGCCATA TGACTAAAGAACCAACTGCCGT-3 ') and a reverse primer (5'- GCTCGAATTCGGATC CAGTAAAGGAGTGAGTGCAAA-3') to amplify the 2,328 bp gene fragment. These primers contain an adapter sequence (underlined) corresponding to the terminal 15 bp sequence of the pET28a fragment cut with NdeI and BamHI restriction enzymes for the In-Fusion reaction. PCR was performed using Q-Cycler (Quanta Biotech, England). PCR amplifications were inserted into pET28a fragments cut with NdeI and BamHI restriction enzymes using an In-Fusion HD cloning kit (Clontech, USA).

1-3. AgaJ5 정제1-3. AgaJ5 Tablets

pHis-AgaJ5를 보유하는 ER2566 세포를 37℃에서 LB 배지에 접종하고 배양한 다음, OD600이 0.5에 도달하였을 때 AgaJ5의 발현을 유도하기 위해 10mM의 isopropyl-β-D-thiogalactopyranoside(IPTG)를 LB 배지에 첨가하고, 16℃에서 12시간 동안 배양하였다. 원심분리하여 세포를 수거하고 lysis buffer(50mM Tris-HCl, 250mM NaCl, pH 7.5)에 재현탁시켰다. 재현탁된 세포를 12,000psi로 French pressure cell을 사용하여 용해시켰다. 세포파편과 상등액을 4℃에서 20분 동안 12,000g로 원심분리하여 분리하였다. BD TALONTM metal affinity resin(Clontech, USA)을 통한 단백질 정제를 위해 상등액만을 사용하였다. Binding buffer로 세척한 후, 250mM 이미다졸을 함유하는 lysis buffer를 사용하여 affinity resin에 결합된 단백질을 용출시켰다. Buffer의 이미다졸을 희석하기 위해, 용출된 시료를 100mM KCl이 함유된 50mM Tris-HCl buffer(pH 8.0)로 4℃에서 10시간 투석하였다. 정제된 AgaJ5의 농도는 Bradford assay를 통해 결정하였다.ER2566 cells harboring pHis-AgaJ5 were inoculated into LB medium at 37 ° C. and cultured. When OD600 reached 0.5, 10 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to LB medium And cultured at 16 DEG C for 12 hours. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, pH 7.5). Resuspended cells were lysed using French pressure cell at 12,000 psi. Cell debris and supernatant were separated by centrifugation at 12,000 g for 20 min at 4 ° C. Only the supernatant was used for protein purification via BD TALON TM metal affinity resin (Clontech, USA). After washing with binding buffer, lysis buffer bound to affinity resin was eluted with lysis buffer containing 250 mM imidazole. To dilute the buffer imidazole, the eluted sample was dialyzed at 50 ° C for 10 hours in 50 mM Tris-HCl buffer (pH 8.0) containing 100 mM KCl. The concentration of purified AgaJ5 was determined by Bradford assay.

1-4. 자이모그램 분석1-4. Self analysis

정제된 AgaJ5를 끓이지 않고 0.1% agarose를 함유하는 12% polyacrylamide gel을 사용하여 분리하였다. 2% Triton X-100에 30분 동안 침지시킨 다음 20mM Tris-HCl buffer(pH 8)로 2회 세척하고, 단백질 겔을 10mM sodium citrate(pH 4.5) 중에서 30℃로 밤새 incubation하였다. AgaJ5의 아가레이즈 활성을 확인하기 위해 Lugol's iodine solution을 사용하여 단백질 겔을 염색하였다.Purified AgaJ5 was isolated using 12% polyacrylamide gel containing 0.1% agarose without boiling. After immersing in 2% Triton X-100 for 30 minutes, the plate was washed twice with 20 mM Tris-HCl buffer (pH 8), and the protein gel was incubated overnight at 30 ° C in 10 mM sodium citrate (pH 4.5). Protein gels were stained with Lugol's iodine solution to confirm agarase activity of AgaJ5.

1-5. 아가레이즈 분석1-5. Agarase analysis

정제된 AgaJ5의 아가레이즈 활성은 이전(Chi et al. 2014.)에 기술된 바와 같이 환원당의 양을 측정하는 3,5-dinitrosalicylic acid(DNS) 방법에 의해 결정하였다. 반응혼합물은 20㎕의 정제된 AgaJ5와 0.1% 아가로오스가 함유된 10mM sodium citrate buffer(pH 4.5) 480㎕로 구성하였다. 30℃에서 30분 동안 incubation한 후, 반응혼합물에 500㎕의 DNS 용액(증류수 100㎖에 DNS 0.65g, 글리세롤 4.5㎖, 2N NaOH 32.5㎖)을 가하였다. 100℃에서 10분간 끓인 후, 환원당의 농도를 측정하기 위해 540nm에서 분광광도계를 사용하여 샘플을 모니터링하였다. 정제된 AgaJ5 대신 20㎕의 증류수를 함유하는 반응혼합물을 대조군으로 사용하였다. AgaJ5 활성의 최적 온도를 결정하기 위해, 10℃와 60℃ 사이의 온도에서 효소 분석을 수행하였다. 이와 유사하게, 최적 pH는 다양한 pH 조건 하에서 효소 반응을 통해 결정하였다 : pH 3, 3.5, 4, 4.5, 5, 5.5 및 6의 10mM sodium citrate buffer, pH 6 및 7의 10mM MOPS buffer, pH 7, 8 및 9의 10mM Tris-HCl buffer, pH 9 및 10의 10mM Glycine-NaOH buffer. 금속 이온의 영향은 NaCl2, MgCl2, CaCl2, ZnCl2, MnCl2, CoCl2, CuCl2, NiCl2, FeCl2, KCl2 및 EDTA와 같은 각 금속 이온을 5mM의 농도로 함유하는 10mM sodium citrate buffer(pH 4.5)에서 효소 반응을 통해 결정하였다.The agarase activity of purified AgaJ5 was determined by the 3,5-dinitrosalicylic acid (DNS) method, which measures the amount of reducing sugar as previously described (Chi et al. 2014.). The reaction mixture consisted of 20 μl of purified AgaJ5 and 480 μl of 10 mM sodium citrate buffer (pH 4.5) containing 0.1% agarose. After incubation at 30 ° C for 30 minutes, 500 μl of a DNS solution (0.60 g of DNS in 100 ml of distilled water, 4.5 ml of glycerol, 32.5 ml of 2N NaOH) was added to the reaction mixture. After boiling at 100 ° C for 10 minutes, the sample was monitored using a spectrophotometer at 540 nm to determine the concentration of reducing sugar. A reaction mixture containing 20 占 퐇 of distilled water instead of purified AgaJ5 was used as a control. To determine the optimal temperature for AgaJ5 activity, enzyme assays were performed at temperatures between 10 < 0 > C and 60 < 0 > C. Similarly, optimal pH was determined by enzyme reaction under various pH conditions: 10 mM sodium citrate buffer at pH 3, 3.5, 4, 4.5, 5, 5.5 and 6, 10 mM MOPS buffer at pH 6 and 7, pH 7, 8 and 9 in 10 mM Tris-HCl buffer, pH 9 and 10 in 10 mM Glycine-NaOH buffer. The effect of the metal ions was evaluated by measuring the concentration of each metal ion such as NaCl 2 , MgCl 2 , CaCl 2 , ZnCl 2 , MnCl 2 , CoCl 2 , CuCl 2 , NiCl 2 , FeCl 2 , KCl 2 and EDTA in 10 mM sodium citrate buffer (pH 4.5).

1-6. 효소 동역학적 분석1-6. Enzyme kinetic analysis

AgaJ5의 동역학적 파라미터(Km 및 Vmax)는 다양한 양의 아가로오스(0.1 ~ 8㎎/㎖)가 함유된 10mM sodium citrate buffer(pH 4.5)에서의 효소반응에 의해 결정하였다. 5% 이하 기질 이용률을 제한하기 위해, 샘플을 30℃에서 7분 동안만 incubation하였다. Km 및 Vmax 값은 Lineweaver-Burk plot으로부터 계산하였다.Kinetic parameters (K m and V max) of AgaJ5 was determined by an enzymatic reaction in agarose (0.1 ~ 8㎎ / ㎖) containing a 10mM sodium citrate buffer (pH 4.5) with varying amounts of agar. To limit the substrate utilization to below 5%, the samples were incubated at 30 ° C for 7 minutes only. The values of K m and V max were calculated from the Lineweaver-Burk plot.

1-7. 점도 측정1-7. Viscosity measurement

AgaJ5의 효소반응은 30℃에서 0.5% 아가로오스가 함유된 10mM sodium citrate buffer(pH 4.5)에서 수행하였다. 반응혼합물의 점도는 DV2T 점도계(Brookfield AMETEK, USA)를 사용하여 0 ~ 60분의 다양한 범위의 반응시간에서 측정하였다.The enzymatic reaction of AgaJ5 was performed at 30 ° C in 10 mM sodium citrate buffer (pH 4.5) containing 0.5% agarose. The viscosity of the reaction mixture was measured using a DV2T viscometer (Brookfield AMETEK, USA) over a range of reaction times ranging from 0 to 60 minutes.

1-8. TLC 분석1-8. TLC analysis

AgaJ5의 효소반응은 0.1% 아가로오스 또는 네오아가로올리고당이 함유된 10mM sodium citrate buffer(pH 4.5)에서 30℃에서 수행하였고, 5분 동안 끓여서 반응을 중단시켰다. Silica Gel 60 plate(Merck, USA)와 n-부탄올 : 아세트산 : H2O 용액(부피 기준 2 : 1 : 1)을 사용하여 반응 샘플의 올리고당을 분리하였다. 20% H2SO4가 함유된 메탄올을 분무한 후, 120℃에서 2분간 가열하여 실리카겔 플레이트 상의 올리고당 스팟(spot)을 가시화하였다. 플레이트는 디지털 카메라 EOS 100D(Canon Inc., Tokyo, Japan)를 사용하여 촬영하였다.The enzymatic reaction of AgaJ5 was performed at 30 ° C in 10 mM sodium citrate buffer (pH 4.5) containing 0.1% agarose or neoagarooligosaccharide, and the reaction was stopped by boiling for 5 minutes. Oligosaccharides of the reaction sample were separated using a Silica Gel 60 plate (Merck, USA) and n-butanol: acetic acid: H 2 O solution (2: 1: 1 by volume). Methanol containing 20% H 2 SO 4 was sprayed and heated at 120 ° C for 2 minutes to visualize an oligosaccharide spot on the silica gel plate. The plate was photographed using a digital camera EOS 100D (Canon Inc., Tokyo, Japan).

1-9. 질량 분광계 분석1-9. Mass spectrometer analysis

가수분해 생성물에 상응하는 TLC 플레이트상의 염색되지 않은 영역을 긁어내어 100% 메탄올에 용해시켰다. 메탄올을 증발시킨 후, matrix-assisted laser desorption ionization time-of-flight mass spectrometer(Autoflex III, Germany)를 사용하여 추출된 올리고당의 분자량을 결정하였다. 매트릭스로 2-(4'-Hydroxybenzeneazo)benzoic acid을 사용하였다.The unstained area on the TLC plate corresponding to the hydrolysis product was scraped off and dissolved in 100% methanol. The molecular weight of the extracted oligosaccharides was determined using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (Autoflex III, Germany) after evaporation of the methanol. 2- (4'-Hydroxybenzeneazo) benzoic acid was used as a matrix.

2. 결과2. Results

2-1. 가야도모나스 주비니에게 G7 유래 새로운 아가레이즈 AgaJ52-1. The new agarazes derived from the G7 to Ganesh Monas Beanie AgaJ5

강한 한천분해능을 갖는 해양박테리아인 G. joobiniege에는 12개의 아가레이즈가 있는 것으로 추정된다. 우리는 그 중에서 두 가지의 새로운 β-아가레이즈인 AgaJ9와 AgaJ11을 특징지었다. AgaJ9는 저온-적응성 아가레이즈 활성을 가지며, AgaJ11은 산성 β-아가레이즈이다. G. joobiniege , a marine bacterium with strong agar resolution, is estimated to have 12 agarases. We have characterized two new β-agarases, AgaJ9 and AgaJ11. AgaJ9 has a low temperature-adaptive agarase activity, and AgaJ11 is an acidic? -Agarase.

G. joobiniege의 새로운 아가레이즈를 연구하기 위해, 805개의 아미노산을 암호화하는 G. joobiniege의 agaJ5 유전자를 클로닝하였다(NCBI Reference Sequence : WP_017446675.1). BLAST를 이용한 비교 서열 분석 결과, 한천분해 해양박테리아인 Catenovulum agarivorans DS-2의 β-아가레이즈(NCBI Reference Sequence : WP_035014943.1)와 AgaJ5가 51%의 동일성(identity)을 보였으며, 한천분해 박테리아인 Cellvibrio sp.의 아가레이즈 AgaA(NCBI Reference Sequence : WP_062064965.1)와 48%의 동일성(64% similarity)을 보였다. 또한 AgaJ5는 Pseudoalteromonas atlantica T6c의 아가레이즈 AgrA(GenBank accession No. AAA25696.1)와 35%의 동일성을 가지며, 심해박테리아인 Microbulbifer thermotolerans JAMB-A94의 β-아가레이즈 AgaO(GenBank accession No. BAK08903.1)와 28%의 동일성을 나타냈다. AgaA, AgrA 및 AgaO 모두 GH86 패밀리에 속하므로, 유사한 AgaJ5도 GH86 패밀리에 속할 것이다. SignalP 프로그램(http://www.cbs.dtu.dk/services/SignalP)은 AgaJ5가 N 말단(아미노산 1 ~ 30)에 시그널 펩티드를 갖는다고 예측했다.To study the new agarase of G. joobiniege, it was cloned gene of G. agaJ5 joobiniege coding for 805 amino acids (NCBI Reference Sequence: WP_017446675.1). Comparison sequence analysis using BLAST showed 51% identity of AgaJ5 with the NCBI Reference Sequence (WP_035014943.1) of Catenovirus agarivorans DS-2, which is an agar-degrading marine bacterium, Showed 48% identity (64% similarity) with Cellvibrio sp. AgaA (NCBI Reference Sequence: WP_062064965.1). In addition, AgaJ5 has 35% identity with AgarA (GenBank accession No. AAA25696.1) of Pseudoalteromonas atlantica T6c, and has a β-agarase AgaO (GenBank accession No. BAK08903.1) of deep-sea bacteria Microbulbifer thermotolerans JAMB- And 28%, respectively. Since both AgaA, AgrA and AgaO belong to the GH86 family, a similar AgaJ5 will also belong to the GH86 family. The SignalP program (http://www.cbs.dtu.dk/services/SignalP) predicts that AgaJ5 has signal peptides at the N-terminal (amino acids 1-30).

AgaJ5의 생화학적 특성을 분석하기 위해, 예상 시그널 펩티드 서열이 없는 성숙한 형태를 암호화하는 agaJ5 유전자 단편을 pET28a 벡터에 삽입하였다. His-태깅 재조합 AgaJ5 단백질을 과발현시키고, TALONTM metal affinity 크로마토그래피를 사용하여 정제하였다(도 1의 A). His-태깅 재조합 AgaJ5의 분자량은 89278Da로 예측되었으며 이는 SDS-PAGE 상의 실험적 분자량과 거의 일치한다(도 1의 A). 정제된 AgaJ5가 아가레이즈 활성을 갖는지 여부를 결정하기 위해 자이모그램 분석을 수행하였다. 정제된 AgaJ5의 활성염색결과 89kDa의 분자량에서 명확한 클리어존(clear zone)을 보였으며(도 1의 B), 이는 AgaJ5가 아가레이즈 활성을 갖는다는 것을 나타낸다.To analyze the biochemical properties of AgaJ5, the agaJ5 gene fragment encoding the mature form without the expected signal peptide sequence was inserted into the pET28a vector. The His-tagging recombinant AgaJ5 protein was over-expressed and purified using TALON ( TM) metal affinity chromatography (Fig. 1 A). The molecular weight of the His-tagged recombinant AgaJ5 was predicted to be 89278 Da, which is almost identical to the experimental molecular weight on SDS-PAGE (Fig. 1 A). A genomic analysis was performed to determine whether purified AgaJ5 had agarase activity. Active staining of the purified AgaJ5 revealed a clear clear zone at a molecular weight of 89 kDa (Fig. 1B), indicating that AgaJ5 has agarase activity.

2-2. pH, 온도 및 금속이온에 따른 AgaJ5의 활성2-2. The activity of AgaJ5 with pH, temperature and metal ion

AgaJ5 아가레이즈 활성의 최적 pH를 결정하기 위해 pH 3 ~ 10 범위 내에서 효소반응을 수행하였다(도 2의 A). AgaJ5의 최대 활성은 pH 4.5에서 관찰되었고, AgaJ5는 4.5 ~ 5.5의 pH 범위에서 최대 효소활성의 80% 이상을 나타내어 산성 아가레이즈임을 시사한다. 유사한 방법으로 10 ~ 60℃의 다양한 온도에서 효소활성을 측정하여 AgaJ5 활성의 최적 온도를 조사한 결과, 최대 활성은 30℃로 관찰되었으며 10℃에서도 40% 이상의 효소활성을 유지하는 것으로 나타났다(도 2의 B). 이러한 저온-적응성 특징은 G. joobiniege의 아가레이즈 AgaJ9와 유사했다. 따라서 이러한 저온-적응성 특징은 G. joobiniege의 아가레이즈의 일반적인 특성일 가능성이 있다.To determine the optimal pH of the AgaJ5 agarase activity, the enzyme reaction was performed in the pH range of 3 to 10 (FIG. 2A). The maximum activity of AgaJ5 was observed at pH 4.5 and AgaJ5 showed more than 80% of maximum enzyme activity in the pH range of 4.5 ~ 5.5, suggesting acid agarase. In a similar manner, the enzyme activity was measured at various temperatures ranging from 10 to 60 ° C, and the optimum activity of AgaJ5 activity was examined. As a result, the maximum activity was observed at 30 ° C and the enzyme activity was maintained at 40% B). This low-temperature-adaptive characteristic was similar to G. joobiniege 's agarase AgaJ9. Thus, these low-temperature-adaptive characteristics are likely to be a general feature of the agarase of G. joobiniege .

또한, 금속이온도 AgaJ5의 아가레이즈 활성에 영향을 미쳤다(도 2의 C). AgaJ5의 효소활성은 CuCl2(1%의 잔여활성), FeCl2(11%의 잔여활성), MnCl2(11%의 잔여활성)와 같은 일부 금속이온에 의해 강하게 억제되는 반면, NaCl 및 KCl과 같은 1가 이온에 의해 약간 활성화되었다. EDTA의 첨가는 AgaJ5의 아가레이즈 활성을 보통의 정도로 억제하였다. 따라서 이러한 결과는 1가 이온이 AgaJ5의 최대 효소활성에 중요하다는 것을 나타낸다. AgaJ5의 Km과 Vmax는 각각 8.9㎎/㎖, 188.6U/㎎이었다(도 3).In addition, the metal affected the agarase activity of the temperature AgaJ5 (Fig. 2C). The enzyme activity of AgaJ5 is strongly inhibited by some metal ions such as CuCl 2 (1% residual activity), FeCl 2 (11% residual activity), MnCl 2 (11% residual activity), while NaCl and KCl It was slightly activated by the same monovalent ions. The addition of EDTA suppressed the agarase activity of AgaJ5 to a normal extent. Thus, these results indicate that monovalent ions are important for maximum enzyme activity of AgaJ5. The K m and V max of AgaJ5 were 8.9 mg / ml and 188.6 U / mg, respectively (Fig. 3).

2-3. AgaJ5의 가수분해물 분석2-3. Analysis of hydrolysates of AgaJ5

AgaJ5가 α- 또는 β-아가레이즈 중 어떤 아가레이즈인지를 조사하기 위해 두 개의 발색 기질인 p-nitrophenyl- -D-galactopyranoside와 p-nitrophenyl- -D-galactopyranoside를 사용하여 AgaJ5의 효소활성을 평가하였다. 생각과 달리 AgaJ5는 어느 기질에서도 활성을 나타내지 않았다. 이것은 G. joobiniege의 다른 아가레이즈인 AgaJ9 및 AgaJ11에서 얻은 결과와 유사하다. AgaJ5가 endo- 또는 exo-타입 중 어떤 타입의 아가레이즈인지를 조사하기 위해, 효소반응혼합물의 점도 변화를 조사하였다. 반응 샘플의 점도는 처음 20분 동안 급격하게 떨어졌으나 반응이 끝날 때까지는 서서히 감소하여 AgaJ5가 endo-타입의 아가레이즈인 것으로 나타났다(도 4의 A).To investigate whether AgaJ5 is an α- or β-agarase, two enzyme substrates, p-nitrophenyl- -D-galactopyranoside and p-nitrophenyl-D-galactopyranoside, were used to evaluate the enzyme activity of AgaJ5 . Unlike the idea, AgaJ5 was not active on any substrate. This is similar to the results obtained with AgaJ9 and AgaJ11, the other agarases of G. joobiniege . To investigate whether AgaJ5 is an endo- or exo- type of agarase, the viscosity change of the enzyme reaction mixture was investigated. The viscosity of the reaction sample dropped sharply during the first 20 minutes but gradually decreased until the end of the reaction, indicating that AgaJ5 was an endo-type agarase (Fig. 4A).

AgaJ5 효소반응의 가수분해생성물을 TLC로 분석하였다(도 4의 B). AgaJ5는 아가로오스를 주로 NA6로 가수분해하였고, 적은 양의 NA2, NA4 및 NA6 보다 큰 네오아가로올리고당을 생산하였다. NA6보다 큰 네오아가로올리고당은 반응 후기 단계에서 사라졌으나, NA2와 NA4는 반응이 끝날 때까지 남았다(도 4의 B). 추가로 NA2, NA4 및 NA6을 기질로 사용하여 TLC 분석을 수행하였다(도 4의 C). 흥미롭게도 밤새 반응시킨 후에도 AgaJ5에 의해 적은 양의 NA2, NA4 및 NA6 만이 가수분해되었으며, 이는 AgaJ5가 NA8보다 작은 네오아가로올리고당은 분해하지 못한다는 것을 의미한다. 이러한 결과는 발색 기질을 이용한 실험에서 AgaJ5 활성이 나타나지 않은 것을 설명하는데 도움이 된다. 이러한 발색 기질은 NA8보다 훨씬 작은 분자이다. TLC 플레이트에서 추출된 올리고당의 분자량을 질량분석기로 조사하였다(도 5 및 6). MALDI-TOF 분석 결과, AgaJ5의 가수분해생성물은 m/z 653(M+Na)+ 및 959(M+Na)+를 나타냈으며, 이는 각각 NA4 및 NA6에 해당한다. 결론적으로 이러한 결과는 AgaJ5가 아가로오스로부터 NA6를 생산하는 endo-타입 β-아가레이즈임을 시사한다.The hydrolysis product of the AgaJ5 enzyme reaction was analyzed by TLC (Fig. 4B). AgaJ5 hydrolyzed agarose to mainly NA6 and produced neoagarooligosaccharides that were smaller than NA2, NA4 and NA6. The neoagarooligosaccharides larger than NA6 disappeared at the later stage of the reaction, but NA2 and NA4 remained until the end of the reaction (Fig. 4B). TLC analysis was further performed using NA2, NA4 and NA6 as substrates (Fig. 4C). Interestingly, even after overnight reaction, only small amounts of NA2, NA4 and NA6 were hydrolyzed by AgaJ5, indicating that AgaJ5 fails to degrade neoagarooligosaccharides smaller than NA8. These results are helpful in explaining the absence of AgaJ5 activity in experiments using chromogenic substrates. These chromogenic substrates are molecules much smaller than NA8. The molecular weight of the oligosaccharides extracted from the TLC plates was investigated using a mass spectrometer (FIGS. 5 and 6). As a result of MALDI-TOF analysis, the hydrolysis products of AgaJ5 showed m / z 653 (M + Na) + and 959 (M + Na) +, which correspond to NA4 and NA6, respectively. In conclusion, these results suggest that AgaJ5 is an endo-type β-agarase producing NA6 from agarose.

<110> Myongji University Industry and Academia Cooperation Foundation National Institute of Biological Resources <120> A beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use thereof <130> PA-D17420 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 775 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 1 Thr Lys Glu Pro Thr Ala Val Ile Asp Thr Ser Pro Asp Arg Phe Thr 1 5 10 15 Leu Asp Ala Ile Thr Gly Ile Ala Leu Asp Thr Trp Val Thr Ser Lys 20 25 30 Pro Phe Lys Val Ala Gly Ile Asn Ala Pro Thr Ala Ile Lys Ile Thr 35 40 45 Asp Gly Glu Tyr Ser Val Asn Gly Ser Ala Phe Thr Asn Gln Ser Gly 50 55 60 Thr Ile Lys Leu Gly Asp Ser Ile Ser Val Arg Ala Lys Ser Lys Ser 65 70 75 80 Glu His Ala Ser Ser His Thr Ala Thr Leu Phe Ile Gly Asp Lys Gln 85 90 95 Ala Asp Phe Ile Ile Thr Thr Leu Ala Ala Pro Thr Phe Asn Gly Thr 100 105 110 Gln Val Asn Val His Leu Asp Thr Leu His Ser Ile Asn Gly Ile Asp 115 120 125 Ser Phe Glu Arg Glu Lys Tyr Ile Thr Ile His Ser Ser Asn Val Glu 130 135 140 Asn Asp Trp Gly Gln Asn Asp Ser His Ser Asn Asn Ala Ala Asn Ala 145 150 155 160 Leu Gly Asp Asp Leu Val Phe Asp Phe Ile Glu Gly His Asn Val Tyr 165 170 175 Phe Gly Arg Glu Thr Gly Ser Leu Gly Trp Asn Leu Arg Asn Val Arg 180 185 190 Gln Asp Asp Ser Arg Pro Gly Tyr Val Asp Pro Ala Ser Leu Ile Ser 195 200 205 Arg Ala Gly Asp Ser Asn Trp Thr Tyr Asp Asn Ser Gln Gln Ala Lys 210 215 220 Phe Lys Asn Gly Arg Gln Leu Glu Asp Arg Met Ala Gly Met Ile Val 225 230 235 240 Gly Gly Gln Gln His Pro Tyr Trp Pro Glu Gly Thr Glu Ile Thr Pro 245 250 255 Val Ala Lys Leu Ala Glu Pro Lys Trp Ser Phe Ser Gln Thr Asp Thr 260 265 270 Ala Asn Glu Pro Leu Gly Thr Ala Thr Gly Glu Tyr Phe Ala Arg Tyr 275 280 285 Leu Gln Asn Phe Tyr Arg Gln Ser Ser Glu Asp Ala Gly Pro Pro Lys 290 295 300 Pro Lys Tyr Phe Glu Val Met Asn Glu Pro Leu Tyr Asp Leu Ser Thr 305 310 315 320 Asp Arg Glu Gly Glu Thr Asn Tyr Val Glu Pro Leu Lys Val Phe Glu 325 330 335 Phe His Asn Thr Val Ala Lys Glu Ile Ala Lys Ile Ser Asp Asn His 340 345 350 Asp Ile Leu Val Gly Gly Tyr Thr Ile Ala Phe Pro Asp Phe Asp Lys 355 360 365 Asn Asn Phe Gln Arg Trp His Asp Arg Asp Lys Leu Phe Ile Asp Thr 370 375 380 Ser Gly Glu Tyr Met Asp Phe Tyr Ser Ile His Leu Tyr Asp Phe Pro 385 390 395 400 Glu Phe Lys Asn Ser Glu Arg Tyr Arg Arg Gly Ser Asn Leu Glu Ala 405 410 415 Thr Leu Asp Met Leu Glu Gln Tyr Ser Gln Ile Lys Leu Gly Glu Ile 420 425 430 Lys Pro Ile Val Ile Ser Glu Val Gly Ser Ser Thr His Ile Met Val 435 440 445 Asn Gln Asp Trp Ser Pro Gln Arg Asp Ala Glu Lys Val Arg Ser Ile 450 455 460 Thr Ser Met Asn Thr Gln Phe Leu Glu Arg Pro Asp Thr Ile Ala Lys 465 470 475 480 Val Ile Pro Phe Ile Pro Val Lys Ala Glu Trp Gly Arg Arg Ile Lys 485 490 495 Asp Asp Asp Thr Ser Ile Ala Tyr Ser Ser Arg Leu Met Ile Gln Gln 500 505 510 Phe Glu Arg Asp Gly Ser Asn Asn Thr Asp Trp Val Tyr Ser Asp His 515 520 525 Ile Tyr Phe Tyr Lys Leu Trp Lys Glu Val Ala Gly Lys Arg Ala Val 530 535 540 Ser Thr Ser Thr Asp Leu Asp Ile Gln Ser Gln Val Phe Ile Asp Gly 545 550 555 560 Lys Thr Ala Tyr Val Val Leu Thr Ser Leu Glu Phe Asp Asp Arg Ala 565 570 575 Ile Gln Leu Asn Thr Leu Gly Val Asp Asn Asn Ser Leu Ser Ser Val 580 585 590 Glu Ile Lys Thr Leu Gly Tyr Ser Glu Gln Asn Thr Val Asp Tyr Gln 595 600 605 Ile Thr Asn Met Thr Glu Leu Pro Asp Asn Trp Val Leu Pro Ala Glu 610 615 620 Ala Thr Gln Ile Ile Lys Leu Thr Tyr Ser Ser Asp Ile Asn Gln Lys 625 630 635 640 Glu Ser Ala Ser Thr Ile Lys Tyr Tyr Ala Ser Glu Tyr Leu Gln Ala 645 650 655 Ile Gln Ala Tyr Ser Pro Met Ser Phe Asn Ile Asp Asp Val Asn Val 660 665 670 Gly Ser Gln Gly Tyr Ala Val Leu Arg Met Gly Leu Gly Arg Asp His 675 680 685 Gly Lys Ser Leu Thr Pro Lys Val Thr Ile Asn Gly Val Thr Leu Thr 690 695 700 Val Pro Ser Asp Phe Arg Gly Tyr Asp Gln Lys Gln Gly Lys Ser Lys 705 710 715 720 Thr Gly Arg Glu Asn Phe Phe Gly Val Ile Glu Ile Pro Val Pro Tyr 725 730 735 Ser Ala Leu Gln Ser Asp Asn Asn Val Gln Ile Glu Phe Ala Asp Asn 740 745 750 Gly Gly Tyr Val Ser Ser Met Ala Leu Gln Val Val Thr Ser Ser Lys 755 760 765 Lys Ile Gln Tyr Thr Leu Asn 770 775 <210> 2 <211> 805 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 2 Met Asn Lys Ala Phe Val Ser Asn Lys Leu Ser Leu Ala Ile Cys Val 1 5 10 15 Ser Leu Phe Leu Thr Ala Cys Gly Gly Gly Gly Ser Thr Glu Thr Lys 20 25 30 Glu Pro Thr Ala Val Ile Asp Thr Ser Pro Asp Arg Phe Thr Leu Asp 35 40 45 Ala Ile Thr Gly Ile Ala Leu Asp Thr Trp Val Thr Ser Lys Pro Phe 50 55 60 Lys Val Ala Gly Ile Asn Ala Pro Thr Ala Ile Lys Ile Thr Asp Gly 65 70 75 80 Glu Tyr Ser Val Asn Gly Ser Ala Phe Thr Asn Gln Ser Gly Thr Ile 85 90 95 Lys Leu Gly Asp Ser Ile Ser Val Arg Ala Lys Ser Lys Ser Glu His 100 105 110 Ala Ser Ser His Thr Ala Thr Leu Phe Ile Gly Asp Lys Gln Ala Asp 115 120 125 Phe Ile Ile Thr Thr Leu Ala Ala Pro Thr Phe Asn Gly Thr Gln Val 130 135 140 Asn Val His Leu Asp Thr Leu His Ser Ile Asn Gly Ile Asp Ser Phe 145 150 155 160 Glu Arg Glu Lys Tyr Ile Thr Ile His Ser Ser Asn Val Glu Asn Asp 165 170 175 Trp Gly Gln Asn Asp Ser His Ser Asn Asn Ala Ala Asn Ala Leu Gly 180 185 190 Asp Asp Leu Val Phe Asp Phe Ile Glu Gly His Asn Val Tyr Phe Gly 195 200 205 Arg Glu Thr Gly Ser Leu Gly Trp Asn Leu Arg Asn Val Arg Gln Asp 210 215 220 Asp Ser Arg Pro Gly Tyr Val Asp Pro Ala Ser Leu Ile Ser Arg Ala 225 230 235 240 Gly Asp Ser Asn Trp Thr Tyr Asp Asn Ser Gln Gln Ala Lys Phe Lys 245 250 255 Asn Gly Arg Gln Leu Glu Asp Arg Met Ala Gly Met Ile Val Gly Gly 260 265 270 Gln Gln His Pro Tyr Trp Pro Glu Gly Thr Glu Ile Thr Pro Val Ala 275 280 285 Lys Leu Ala Glu Pro Lys Trp Ser Phe Ser Gln Thr Asp Thr Ala Asn 290 295 300 Glu Pro Leu Gly Thr Ala Thr Gly Glu Tyr Phe Ala Arg Tyr Leu Gln 305 310 315 320 Asn Phe Tyr Arg Gln Ser Ser Glu Asp Ala Gly Pro Pro Lys Pro Lys 325 330 335 Tyr Phe Glu Val Met Asn Glu Pro Leu Tyr Asp Leu Ser Thr Asp Arg 340 345 350 Glu Gly Glu Thr Asn Tyr Val Glu Pro Leu Lys Val Phe Glu Phe His 355 360 365 Asn Thr Val Ala Lys Glu Ile Ala Lys Ile Ser Asp Asn His Asp Ile 370 375 380 Leu Val Gly Gly Tyr Thr Ile Ala Phe Pro Asp Phe Asp Lys Asn Asn 385 390 395 400 Phe Gln Arg Trp His Asp Arg Asp Lys Leu Phe Ile Asp Thr Ser Gly 405 410 415 Glu Tyr Met Asp Phe Tyr Ser Ile His Leu Tyr Asp Phe Pro Glu Phe 420 425 430 Lys Asn Ser Glu Arg Tyr Arg Arg Gly Ser Asn Leu Glu Ala Thr Leu 435 440 445 Asp Met Leu Glu Gln Tyr Ser Gln Ile Lys Leu Gly Glu Ile Lys Pro 450 455 460 Ile Val Ile Ser Glu Val Gly Ser Ser Thr His Ile Met Val Asn Gln 465 470 475 480 Asp Trp Ser Pro Gln Arg Asp Ala Glu Lys Val Arg Ser Ile Thr Ser 485 490 495 Met Asn Thr Gln Phe Leu Glu Arg Pro Asp Thr Ile Ala Lys Val Ile 500 505 510 Pro Phe Ile Pro Val Lys Ala Glu Trp Gly Arg Arg Ile Lys Asp Asp 515 520 525 Asp Thr Ser Ile Ala Tyr Ser Ser Arg Leu Met Ile Gln Gln Phe Glu 530 535 540 Arg Asp Gly Ser Asn Asn Thr Asp Trp Val Tyr Ser Asp His Ile Tyr 545 550 555 560 Phe Tyr Lys Leu Trp Lys Glu Val Ala Gly Lys Arg Ala Val Ser Thr 565 570 575 Ser Thr Asp Leu Asp Ile Gln Ser Gln Val Phe Ile Asp Gly Lys Thr 580 585 590 Ala Tyr Val Val Leu Thr Ser Leu Glu Phe Asp Asp Arg Ala Ile Gln 595 600 605 Leu Asn Thr Leu Gly Val Asp Asn Asn Ser Leu Ser Ser Val Glu Ile 610 615 620 Lys Thr Leu Gly Tyr Ser Glu Gln Asn Thr Val Asp Tyr Gln Ile Thr 625 630 635 640 Asn Met Thr Glu Leu Pro Asp Asn Trp Val Leu Pro Ala Glu Ala Thr 645 650 655 Gln Ile Ile Lys Leu Thr Tyr Ser Ser Asp Ile Asn Gln Lys Glu Ser 660 665 670 Ala Ser Thr Ile Lys Tyr Tyr Ala Ser Glu Tyr Leu Gln Ala Ile Gln 675 680 685 Ala Tyr Ser Pro Met Ser Phe Asn Ile Asp Asp Val Asn Val Gly Ser 690 695 700 Gln Gly Tyr Ala Val Leu Arg Met Gly Leu Gly Arg Asp His Gly Lys 705 710 715 720 Ser Leu Thr Pro Lys Val Thr Ile Asn Gly Val Thr Leu Thr Val Pro 725 730 735 Ser Asp Phe Arg Gly Tyr Asp Gln Lys Gln Gly Lys Ser Lys Thr Gly 740 745 750 Arg Glu Asn Phe Phe Gly Val Ile Glu Ile Pro Val Pro Tyr Ser Ala 755 760 765 Leu Gln Ser Asp Asn Asn Val Gln Ile Glu Phe Ala Asp Asn Gly Gly 770 775 780 Tyr Val Ser Ser Met Ala Leu Gln Val Val Thr Ser Ser Lys Lys Ile 785 790 795 800 Gln Tyr Thr Leu Asn 805 <210> 3 <211> 796 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 3 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Thr Lys Glu Pro Thr Ala Val Ile Asp Thr Ser 20 25 30 Pro Asp Arg Phe Thr Leu Asp Ala Ile Thr Gly Ile Ala Leu Asp Thr 35 40 45 Trp Val Thr Ser Lys Pro Phe Lys Val Ala Gly Ile Asn Ala Pro Thr 50 55 60 Ala Ile Lys Ile Thr Asp Gly Glu Tyr Ser Val Asn Gly Ser Ala Phe 65 70 75 80 Thr Asn Gln Ser Gly Thr Ile Lys Leu Gly Asp Ser Ile Ser Val Arg 85 90 95 Ala Lys Ser Lys Ser Glu His Ala Ser Ser His Thr Ala Thr Leu Phe 100 105 110 Ile Gly Asp Lys Gln Ala Asp Phe Ile Ile Thr Thr Leu Ala Ala Pro 115 120 125 Thr Phe Asn Gly Thr Gln Val Asn Val His Leu Asp Thr Leu His Ser 130 135 140 Ile Asn Gly Ile Asp Ser Phe Glu Arg Glu Lys Tyr Ile Thr Ile His 145 150 155 160 Ser Ser Asn Val Glu Asn Asp Trp Gly Gln Asn Asp Ser His Ser Asn 165 170 175 Asn Ala Ala Asn Ala Leu Gly Asp Asp Leu Val Phe Asp Phe Ile Glu 180 185 190 Gly His Asn Val Tyr Phe Gly Arg Glu Thr Gly Ser Leu Gly Trp Asn 195 200 205 Leu Arg Asn Val Arg Gln Asp Asp Ser Arg Pro Gly Tyr Val Asp Pro 210 215 220 Ala Ser Leu Ile Ser Arg Ala Gly Asp Ser Asn Trp Thr Tyr Asp Asn 225 230 235 240 Ser Gln Gln Ala Lys Phe Lys Asn Gly Arg Gln Leu Glu Asp Arg Met 245 250 255 Ala Gly Met Ile Val Gly Gly Gln Gln His Pro Tyr Trp Pro Glu Gly 260 265 270 Thr Glu Ile Thr Pro Val Ala Lys Leu Ala Glu Pro Lys Trp Ser Phe 275 280 285 Ser Gln Thr Asp Thr Ala Asn Glu Pro Leu Gly Thr Ala Thr Gly Glu 290 295 300 Tyr Phe Ala Arg Tyr Leu Gln Asn Phe Tyr Arg Gln Ser Ser Glu Asp 305 310 315 320 Ala Gly Pro Pro Lys Pro Lys Tyr Phe Glu Val Met Asn Glu Pro Leu 325 330 335 Tyr Asp Leu Ser Thr Asp Arg Glu Gly Glu Thr Asn Tyr Val Glu Pro 340 345 350 Leu Lys Val Phe Glu Phe His Asn Thr Val Ala Lys Glu Ile Ala Lys 355 360 365 Ile Ser Asp Asn His Asp Ile Leu Val Gly Gly Tyr Thr Ile Ala Phe 370 375 380 Pro Asp Phe Asp Lys Asn Asn Phe Gln Arg Trp His Asp Arg Asp Lys 385 390 395 400 Leu Phe Ile Asp Thr Ser Gly Glu Tyr Met Asp Phe Tyr Ser Ile His 405 410 415 Leu Tyr Asp Phe Pro Glu Phe Lys Asn Ser Glu Arg Tyr Arg Arg Gly 420 425 430 Ser Asn Leu Glu Ala Thr Leu Asp Met Leu Glu Gln Tyr Ser Gln Ile 435 440 445 Lys Leu Gly Glu Ile Lys Pro Ile Val Ile Ser Glu Val Gly Ser Ser 450 455 460 Thr His Ile Met Val Asn Gln Asp Trp Ser Pro Gln Arg Asp Ala Glu 465 470 475 480 Lys Val Arg Ser Ile Thr Ser Met Asn Thr Gln Phe Leu Glu Arg Pro 485 490 495 Asp Thr Ile Ala Lys Val Ile Pro Phe Ile Pro Val Lys Ala Glu Trp 500 505 510 Gly Arg Arg Ile Lys Asp Asp Asp Thr Ser Ile Ala Tyr Ser Ser Arg 515 520 525 Leu Met Ile Gln Gln Phe Glu Arg Asp Gly Ser Asn Asn Thr Asp Trp 530 535 540 Val Tyr Ser Asp His Ile Tyr Phe Tyr Lys Leu Trp Lys Glu Val Ala 545 550 555 560 Gly Lys Arg Ala Val Ser Thr Ser Thr Asp Leu Asp Ile Gln Ser Gln 565 570 575 Val Phe Ile Asp Gly Lys Thr Ala Tyr Val Val Leu Thr Ser Leu Glu 580 585 590 Phe Asp Asp Arg Ala Ile Gln Leu Asn Thr Leu Gly Val Asp Asn Asn 595 600 605 Ser Leu Ser Ser Val Glu Ile Lys Thr Leu Gly Tyr Ser Glu Gln Asn 610 615 620 Thr Val Asp Tyr Gln Ile Thr Asn Met Thr Glu Leu Pro Asp Asn Trp 625 630 635 640 Val Leu Pro Ala Glu Ala Thr Gln Ile Ile Lys Leu Thr Tyr Ser Ser 645 650 655 Asp Ile Asn Gln Lys Glu Ser Ala Ser Thr Ile Lys Tyr Tyr Ala Ser 660 665 670 Glu Tyr Leu Gln Ala Ile Gln Ala Tyr Ser Pro Met Ser Phe Asn Ile 675 680 685 Asp Asp Val Asn Val Gly Ser Gln Gly Tyr Ala Val Leu Arg Met Gly 690 695 700 Leu Gly Arg Asp His Gly Lys Ser Leu Thr Pro Lys Val Thr Ile Asn 705 710 715 720 Gly Val Thr Leu Thr Val Pro Ser Asp Phe Arg Gly Tyr Asp Gln Lys 725 730 735 Gln Gly Lys Ser Lys Thr Gly Arg Glu Asn Phe Phe Gly Val Ile Glu 740 745 750 Ile Pro Val Pro Tyr Ser Ala Leu Gln Ser Asp Asn Asn Val Gln Ile 755 760 765 Glu Phe Ala Asp Asn Gly Gly Tyr Val Ser Ser Met Ala Leu Gln Val 770 775 780 Val Thr Ser Ser Lys Lys Ile Gln Tyr Thr Leu Asn 785 790 795 <210> 4 <211> 2328 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 4 actaaagaac caactgccgt tatcgatacc agccctgacc gctttaccct tgacgcaata 60 acaggcattg ctttagatac ttgggtaaca agcaaaccct ttaaagtagc aggtattaat 120 gcgccgaccg caataaaaat aacggatgga gaatactctg ttaatggctc agcttttact 180 aatcaaagcg gcactataaa gctgggcgat agcatatctg tcagagctaa aagcaaatct 240 gagcacgcta gttcccatac cgccacttta ttcattggcg ataaacaagc agactttatt 300 atcaccacct tagcggcccc tacgtttaat ggcacccagg tcaatgtgca tttagatacc 360 ttgcacagca taaatggcat tgatagtttt gagcgcgaaa aatacatcac aatacattca 420 agcaacgtcg aaaatgactg gggacaaaac gatagccata gtaacaatgc tgccaatgca 480 ttaggtgatg acttagtgtt tgattttatt gaaggccata acgtttattt tggacgtgaa 540 actggtagct tgggctggaa tttacgcaat gttcgccaag atgatagccg acccggttat 600 gttgaccctg ccagtctaat aagccgcgcg ggcgattcta actggacata cgacaacagc 660 cagcaagcta aatttaaaaa tggccgtcaa cttgaagatc gcatggccgg aatgatagtt 720 ggtggccaac agcaccctta ttggccagaa ggcacagaga tcacgccagt ggcaaaatta 780 gctgagccaa aatggtcatt ttcacagaca gatactgcta atgaaccatt aggaactgcc 840 accggagaat attttgctcg atatttgcaa aacttttacc gtcaatcatc agaagatgct 900 gggcccccta aacctaaata ctttgaagtg atgaacgaac ctttatacga tttaagtaca 960 gacagagaag gtgaaacaaa ttatgtggag ccgctaaaag tctttgagtt tcataatact 1020 gtggccaaag agatagctaa aattagtgac aaccatgata ttttagtggg aggttatacg 1080 atagcttttc cagattttga taaaaacaac tttcagcgct ggcatgacag agacaaattg 1140 tttattgata catccggcga atacatggat ttttattcaa tccatttgta cgattttcct 1200 gagtttaaaa actcagagcg ttatcgccgt ggcagtaatt tagaagccac tttagatatg 1260 ttagagcaat atagccaaat aaaacttggg gaaattaaac caattgtaat ttccgaagtg 1320 ggctcatcga ctcatatcat ggttaaccaa gattggagcc cgcaaaggga tgccgaaaag 1380 gtacgctcaa ttaccagcat gaatactcag tttttagagc gtcccgatac gatagctaaa 1440 gtgatccctt ttattcctgt gaaagcagaa tggggtcgtc gaataaaaga tgatgatacc 1500 agcatcgcct attcgagccg tttgatgatc caacagtttg agcgtgatgg cagcaataat 1560 acagactggg tttattctga tcatatttat ttttacaaac tatggaaaga ggtagctggt 1620 aaacgcgccg ttagcacaag cacagactta gatattcaaa gccaagtatt tattgatggt 1680 aaaacggctt atgtggtgtt aaccagctta gagtttgatg atagagcaat ccagttgaat 1740 actttaggcg ttgataataa ttcattaagt tcggttgaaa tcaaaacgct tggttatagt 1800 gagcaaaata cagttgatta tcaaatcact aatatgactg agttgcctga taattgggtt 1860 ttacctgccg aagccactca aattatcaaa ctcacttata gcagtgatat caatcaaaaa 1920 gaatcagcga gtacaattaa atattatgct agtgagtact tacaggcaat acaagcctat 1980 agtccaatga gttttaacat cgatgatgtg aatgttggca gtcaaggata tgctgtgcta 2040 agaatgggtt tagggcgcga tcatggtaaa tccttaacac caaaagtaac aattaatggt 2100 gtcacgctaa ccgtacccag tgattttaga ggttatgatc aaaaacaagg gaaatcaaaa 2160 actggccgcg aaaacttttt tggtgtgata gagatccctg tgccttactc agcactgcaa 2220 agcgataata atgttcagat tgagtttgct gacaacggcg gttatgtcag tagtatggcg 2280 ctgcaagtgg tcactagcag taaaaagatt caatatacac ttaattaa 2328 <210> 5 <211> 2418 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 5 atgaataaag cctttgtgag caataaacta agtttagcga tttgtgtaag ccttttttta 60 acggcctgtg gtggtggcgg ttcgacagaa actaaagaac caactgccgt tatcgatacc 120 agccctgacc gctttaccct tgacgcaata acaggcattg ctttagatac ttgggtaaca 180 agcaaaccct ttaaagtagc aggtattaat gcgccgaccg caataaaaat aacggatgga 240 gaatactctg ttaatggctc agcttttact aatcaaagcg gcactataaa gctgggcgat 300 agcatatctg tcagagctaa aagcaaatct gagcacgcta gttcccatac cgccacttta 360 ttcattggcg ataaacaagc agactttatt atcaccacct tagcggcccc tacgtttaat 420 ggcacccagg tcaatgtgca tttagatacc ttgcacagca taaatggcat tgatagtttt 480 gagcgcgaaa aatacatcac aatacattca agcaacgtcg aaaatgactg gggacaaaac 540 gatagccata gtaacaatgc tgccaatgca ttaggtgatg acttagtgtt tgattttatt 600 gaaggccata acgtttattt tggacgtgaa actggtagct tgggctggaa tttacgcaat 660 gttcgccaag atgatagccg acccggttat gttgaccctg ccagtctaat aagccgcgcg 720 ggcgattcta actggacata cgacaacagc cagcaagcta aatttaaaaa tggccgtcaa 780 cttgaagatc gcatggccgg aatgatagtt ggtggccaac agcaccctta ttggccagaa 840 ggcacagaga tcacgccagt ggcaaaatta gctgagccaa aatggtcatt ttcacagaca 900 gatactgcta atgaaccatt aggaactgcc accggagaat attttgctcg atatttgcaa 960 aacttttacc gtcaatcatc agaagatgct gggcccccta aacctaaata ctttgaagtg 1020 atgaacgaac ctttatacga tttaagtaca gacagagaag gtgaaacaaa ttatgtggag 1080 ccgctaaaag tctttgagtt tcataatact gtggccaaag agatagctaa aattagtgac 1140 aaccatgata ttttagtggg aggttatacg atagcttttc cagattttga taaaaacaac 1200 tttcagcgct ggcatgacag agacaaattg tttattgata catccggcga atacatggat 1260 ttttattcaa tccatttgta cgattttcct gagtttaaaa actcagagcg ttatcgccgt 1320 ggcagtaatt tagaagccac tttagatatg ttagagcaat atagccaaat aaaacttggg 1380 gaaattaaac caattgtaat ttccgaagtg ggctcatcga ctcatatcat ggttaaccaa 1440 gattggagcc cgcaaaggga tgccgaaaag gtacgctcaa ttaccagcat gaatactcag 1500 tttttagagc gtcccgatac gatagctaaa gtgatccctt ttattcctgt gaaagcagaa 1560 tggggtcgtc gaataaaaga tgatgatacc agcatcgcct attcgagccg tttgatgatc 1620 caacagtttg agcgtgatgg cagcaataat acagactggg tttattctga tcatatttat 1680 ttttacaaac tatggaaaga ggtagctggt aaacgcgccg ttagcacaag cacagactta 1740 gatattcaaa gccaagtatt tattgatggt aaaacggctt atgtggtgtt aaccagctta 1800 gagtttgatg atagagcaat ccagttgaat actttaggcg ttgataataa ttcattaagt 1860 tcggttgaaa tcaaaacgct tggttatagt gagcaaaata cagttgatta tcaaatcact 1920 aatatgactg agttgcctga taattgggtt ttacctgccg aagccactca aattatcaaa 1980 ctcacttata gcagtgatat caatcaaaaa gaatcagcga gtacaattaa atattatgct 2040 agtgagtact tacaggcaat acaagcctat agtccaatga gttttaacat cgatgatgtg 2100 aatgttggca gtcaaggata tgctgtgcta agaatgggtt tagggcgcga tcatggtaaa 2160 tccttaacac caaaagtaac aattaatggt gtcacgctaa ccgtacccag tgattttaga 2220 ggttatgatc aaaaacaagg gaaatcaaaa actggccgcg aaaacttttt tggtgtgata 2280 gagatccctg tgccttactc agcactgcaa agcgataata atgttcagat tgagtttgct 2340 gacaacggcg gttatgtcag tagtatggcg ctgcaagtgg tcactagcag taaaaagatt 2400 caatatacac ttaattaa 2418 <210> 6 <211> 2391 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 6 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgactaaag aaccaactgc cgttatcgat accagccctg accgctttac ccttgacgca 120 ataacaggca ttgctttaga tacttgggta acaagcaaac cctttaaagt agcaggtatt 180 aatgcgccga ccgcaataaa aataacggat ggagaatact ctgttaatgg ctcagctttt 240 actaatcaaa gcggcactat aaagctgggc gatagcatat ctgtcagagc taaaagcaaa 300 tctgagcacg ctagttccca taccgccact ttattcattg gcgataaaca agcagacttt 360 attatcacca ccttagcggc ccctacgttt aatggcaccc aggtcaatgt gcatttagat 420 accttgcaca gcataaatgg cattgatagt tttgagcgcg aaaaatacat cacaatacat 480 tcaagcaacg tcgaaaatga ctggggacaa aacgatagcc atagtaacaa tgctgccaat 540 gcattaggtg atgacttagt gtttgatttt attgaaggcc ataacgttta ttttggacgt 600 gaaactggta gcttgggctg gaatttacgc aatgttcgcc aagatgatag ccgacccggt 660 tatgttgacc ctgccagtct aataagccgc gcgggcgatt ctaactggac atacgacaac 720 agccagcaag ctaaatttaa aaatggccgt caacttgaag atcgcatggc cggaatgata 780 gttggtggcc aacagcaccc ttattggcca gaaggcacag agatcacgcc agtggcaaaa 840 ttagctgagc caaaatggtc attttcacag acagatactg ctaatgaacc attaggaact 900 gccaccggag aatattttgc tcgatatttg caaaactttt accgtcaatc atcagaagat 960 gctgggcccc ctaaacctaa atactttgaa gtgatgaacg aacctttata cgatttaagt 1020 acagacagag aaggtgaaac aaattatgtg gagccgctaa aagtctttga gtttcataat 1080 actgtggcca aagagatagc taaaattagt gacaaccatg atattttagt gggaggttat 1140 acgatagctt ttccagattt tgataaaaac aactttcagc gctggcatga cagagacaaa 1200 ttgtttattg atacatccgg cgaatacatg gatttttatt caatccattt gtacgatttt 1260 cctgagttta aaaactcaga gcgttatcgc cgtggcagta atttagaagc cactttagat 1320 atgttagagc aatatagcca aataaaactt ggggaaatta aaccaattgt aatttccgaa 1380 gtgggctcat cgactcatat catggttaac caagattgga gcccgcaaag ggatgccgaa 1440 aaggtacgct caattaccag catgaatact cagtttttag agcgtcccga tacgatagct 1500 aaagtgatcc cttttattcc tgtgaaagca gaatggggtc gtcgaataaa agatgatgat 1560 accagcatcg cctattcgag ccgtttgatg atccaacagt ttgagcgtga tggcagcaat 1620 aatacagact gggtttattc tgatcatatt tatttttaca aactatggaa agaggtagct 1680 ggtaaacgcg ccgttagcac aagcacagac ttagatattc aaagccaagt atttattgat 1740 ggtaaaacgg cttatgtggt gttaaccagc ttagagtttg atgatagagc aatccagttg 1800 aatactttag gcgttgataa taattcatta agttcggttg aaatcaaaac gcttggttat 1860 agtgagcaaa atacagttga ttatcaaatc actaatatga ctgagttgcc tgataattgg 1920 gttttacctg ccgaagccac tcaaattatc aaactcactt atagcagtga tatcaatcaa 1980 aaagaatcag cgagtacaat taaatattat gctagtgagt acttacaggc aatacaagcc 2040 tatagtccaa tgagttttaa catcgatgat gtgaatgttg gcagtcaagg atatgctgtg 2100 ctaagaatgg gtttagggcg cgatcatggt aaatccttaa caccaaaagt aacaattaat 2160 ggtgtcacgc taaccgtacc cagtgatttt agaggttatg atcaaaaaca agggaaatca 2220 aaaactggcc gcgaaaactt ttttggtgtg atagagatcc ctgtgcctta ctcagcactg 2280 caaagcgata ataatgttca gattgagttt gctgacaacg gcggttatgt cagtagtatg 2340 gcgctgcaag tggtcactag cagtaaaaag attcaatata cacttaatta a 2391 <110> Myongji University Industry and Academia Cooperation Foundation National Institute of Biological Resources <120> A beta-agarase AgaJ5 from Gayadomonas joobiniege G7 and use          the <130> PA-D17420 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 775 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 1 Thr Lys Glu Pro Thr Ala Val Ile Asp Thr Ser Pro Asp Arg Phe Thr   1 5 10 15 Leu Asp Ala Ile Thr Gly Ile Ala Leu Asp Thr Trp Val Thr Ser Lys              20 25 30 Pro Phe Lys Val Ala Gly Ile Asn Ala Pro Thr Ala Ile Lys Ile Thr          35 40 45 Asp Gly Glu Tyr Ser Val Asn Gly Ser Ala Phe Thr Asn Gln Ser Gly      50 55 60 Thr Ile Lys Leu Gly Asp Ser Ile Ser Val Arg Ala Lys Ser Lys Ser  65 70 75 80 Glu His Ala Ser Ser His Thr Ala Thr Leu Phe Ile Gly Asp Lys Gln                  85 90 95 Ala Asp Phe Ile Ile Thr Thr Leu Ala Ala Pro Thr Phe Asn Gly Thr             100 105 110 Gln Val Asn Val His Leu Asp Thr Leu His Ser Ile Asn Gly Ile Asp         115 120 125 Ser Phe Glu Arg Glu Lys Tyr Ile Thr Ile His Ser Ser Asn Val Glu     130 135 140 Asn Asp Trp Gly Gln Asn Asp Ser His Ser Asn Asn Ala Ala Asn Ala 145 150 155 160 Leu Gly Asp Asp Leu Val Phe Asp Phe Ile Glu Gly His Asn Val Tyr                 165 170 175 Phe Gly Arg Glu Thr Gly Ser Leu Gly Trp Asn Leu Arg Asn Val Arg             180 185 190 Gln Asp Asp Ser Arg Pro Gly Tyr Val Asp Pro Ala Ser Leu Ile Ser         195 200 205 Arg Ala Gly Asp Ser Asn Trp Thr Tyr Asp Asn Ser Gln Gln Ala Lys     210 215 220 Phe Lys Asn Gly Arg Gln Leu Glu Asp Arg Met Ala Gly Met Ile Val 225 230 235 240 Gly Gly Gln Gln His Pro Tyr Trp Pro Glu Gly Thr Glu Ile Thr Pro                 245 250 255 Val Ala Lys Leu Ala Glu Pro Lys Trp Ser Phe Ser Gln Thr Asp Thr             260 265 270 Ala Asn Glu Pro Leu Gly Thr Ala Thr Gly Glu Tyr Phe Ala Arg Tyr         275 280 285 Leu Gln Asn Phe Tyr Arg Gln Ser Ser Glu Asp Ala Gly Pro Pro Lys     290 295 300 Pro Lys Tyr Phe Glu Val Met Asn Glu Pro Leu Tyr Asp Leu Ser Thr 305 310 315 320 Asp Arg Glu Gly Glu Thr Asn Tyr Val Glu Pro Leu Lys Val Phe Glu                 325 330 335 Phe His Asn Thr Val Ala Lys Glu Ile Ala Lys Ile Ser Asp Asn His             340 345 350 Asp Ile Leu Val Gly Gly Tyr Thr Ile Ala Phe Pro Asp Phe Asp Lys         355 360 365 Asn Asn Phe Gln Arg Trp His Asp Arg Asp Lys Leu Phe Ile Asp Thr     370 375 380 Ser Gly Glu Tyr Met Asp Phe Tyr Ser Ile His Leu Tyr Asp Phe Pro 385 390 395 400 Glu Phe Lys Asn Ser Glu Arg Tyr Arg Arg Gly Ser Asn Leu Glu Ala                 405 410 415 Thr Leu Asp Met Leu Glu Gln Tyr Ser Gln Ile Lys Leu Gly Glu Ile             420 425 430 Lys Pro Ile Val Ile Ser Glu Val Gly Ser Ser Thr His Ile Met Val         435 440 445 Asn Gln Asp Trp Ser Pro Gln Arg Asp Ala Glu Lys Val Arg Ser Ile     450 455 460 Thr Ser Met Asn Thr Gln Phe Leu Glu Arg Pro Asp Thr Ile Ala Lys 465 470 475 480 Val Ile Pro Phe Ile Pro Val Lys Ala Glu Trp Gly Arg Arg Ile Lys                 485 490 495 Asp Asp Thr Ser Ile Ala Tyr Ser Ser Arg Leu Met Ile Gln Gln             500 505 510 Phe Glu Arg Asp Gly Ser Asn Asn Thr Asp Trp Val Tyr Ser Asp His         515 520 525 Ile Tyr Phe Tyr Lys Leu Trp Lys Glu Val Ala Gly Lys Arg Ala Val     530 535 540 Ser Thr Ser Thr Asp Leu Asp Ile Gln Ser Gln Val Phe Ile Asp Gly 545 550 555 560 Lys Thr Ala Tyr Val Val Leu Thr Ser Leu Glu Phe Asp Asp Arg Ala                 565 570 575 Ile Gln Leu Asn Thr Leu Gly Val Asp Asn Asn Ser Leu Ser Ser Val             580 585 590 Glu Ile Lys Thr Leu Gly Tyr Ser Glu Gln Asn Thr Val Asp Tyr Gln         595 600 605 Ile Thr Asn Met Thr Glu Leu Pro Asp Asn Trp Val Leu Pro Ala Glu     610 615 620 Ala Thr Gln Ile Ile Lys Leu Thr Tyr Ser Ser Asp Ile Asn Gln Lys 625 630 635 640 Glu Ser Ala Ser Thr Ile Lys Tyr Tyr Ala Ser Glu Tyr Leu Gln Ala                 645 650 655 Ile Gln Ala Tyr Ser Pro Met Ser Phe Asn Ile Asp Asp Val Asn Val             660 665 670 Gly Ser Gln Gly Tyr Ala Val Leu Arg Met Gly Leu Gly Arg Asp His         675 680 685 Gly Lys Ser Leu Thr Pro Lys Val Thr Ile Asn Gly Val Thr Leu Thr     690 695 700 Val Pro Ser Asp Phe Arg Gly Tyr Asp Gln Lys Gln Gly Lys Ser Lys 705 710 715 720 Thr Gly Arg Glu Asn Phe Phe Gly Val Ile Glu Ile Pro Val Pro Tyr                 725 730 735 Ser Ala Leu Gln Ser Asp Asn Asn Val Gln Ile Glu Phe Ala Asp Asn             740 745 750 Gly Gly Tyr Val Ser Ser Met Ala Leu Gln Val Val Thr Ser Ser Lys         755 760 765 Lys Ile Gln Tyr Thr Leu Asn     770 775 <210> 2 <211> 805 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 2 Met Asn Lys Ala Phe Val Ser Asn Lys Leu Ser Leu Ala Ile Cys Val   1 5 10 15 Ser Leu Phe Leu Thr Ala Cys Gly Gly Gly Gly Ser Thr Glu Thr Lys              20 25 30 Glu Pro Thr Ala Val Ile Asp Thr Ser Pro Asp Arg Phe Thr Leu Asp          35 40 45 Ala Ile Thr Gly Ile Ala Leu Asp Thr Trp Val Thr Ser Lys Pro Phe      50 55 60 Lys Val Ala Gly Ile Asn Ala Pro Thr Ala Ile Lys Ile Thr Asp Gly  65 70 75 80 Glu Tyr Ser Val Asn Gly Ser Ala Phe Thr Asn Gln Ser Gly Thr Ile                  85 90 95 Lys Leu Gly Asp Ser Ile Ser Val Arg Ala Lys Ser Lys Ser Glu His             100 105 110 Ala Ser Ser His Thr Ala Thr Leu Phe Ile Gly Asp Lys Gln Ala Asp         115 120 125 Phe Ile Ile Thr Thr Leu Ala Ala Pro Thr Phe Asn Gly Thr Gln Val     130 135 140 Asn Val His Leu Asp Thr Leu His Ser Ile Asn Gly Ile Asp Ser Phe 145 150 155 160 Glu Arg Glu Lys Tyr Ile Thr Ile His Ser Ser Asn Val Glu Asn Asp                 165 170 175 Trp Gly Gln Asn Asp Ser His Ser Asn Asn Ala Ala Asn Ala Leu Gly             180 185 190 Asp Asp Leu Val Phe Asp Phe Ile Glu Gly His Asn Val Tyr Phe Gly         195 200 205 Arg Glu Thr Gly Ser Leu Gly Trp Asn Leu Arg Asn Val Arg Gln Asp     210 215 220 Asp Ser Arg Pro Gly Tyr Val Asp Pro Ala Ser Leu Ile Ser Arg Ala 225 230 235 240 Gly Asp Ser Asn Trp Thr Tyr Asp Asn Ser Gln Gln Ala Lys Phe Lys                 245 250 255 Asn Gly Arg Gln Leu Glu Asp Arg Met Ala Gly Met Ile Val Gly Gly             260 265 270 Gln Gln His Pro Tyr Trp Pro Glu Gly Thr Glu Ile Thr Pro Val Ala         275 280 285 Lys Leu Ala Glu Pro Lys Trp Ser Phe Ser Gln Thr Asp Thr Ala Asn     290 295 300 Glu Pro Leu Gly Thr Ala Thr Gly Glu Tyr Phe Ala Arg Tyr Leu Gln 305 310 315 320 Asn Phe Tyr Arg Gln Ser Ser Glu Asp Ala Gly Pro Pro Lys Pro Lys                 325 330 335 Tyr Phe Glu Val Met Asn Glu Pro Leu Tyr Asp Leu Ser Thr Asp Arg             340 345 350 Glu Gly Glu Thr Asn Tyr Val Glu Pro Leu Lys Val Phe Glu Phe His         355 360 365 Asn Thr Val Ala Lys Glu Ile Ala Lys Ile Ser Asp Asn His Asp Ile     370 375 380 Leu Val Gly Gly Tyr Thr Ile Ala Phe Pro Asp Phe Asp Lys Asn Asn 385 390 395 400 Phe Gln Arg Trp His Asp Arg Asp Lys Leu Phe Ile Asp Thr Ser Gly                 405 410 415 Glu Tyr Met Asp Phe Tyr Ser Ile His Leu Tyr Asp Phe Pro Glu Phe             420 425 430 Lys Asn Ser Glu Arg Tyr Arg Arg Gly Ser Asn Leu Glu Ala Thr Leu         435 440 445 Asp Met Leu Glu Gln Tyr Ser Gln Ile Lys Leu Gly Glu Ile Lys Pro     450 455 460 Ile Val Ile Ser Glu Val Gly Ser Ser Thr His Ile Met Val Asn Gln 465 470 475 480 Asp Trp Ser Pro Gln Arg Asp Ala Glu Lys Val Arg Ser Ser Thr Ser                 485 490 495 Met Asn Thr Gln Phe Leu Glu Arg Pro Asp Thr Ile Ala Lys Val Ile             500 505 510 Pro Phe Ile Pro Val Lys Ala Glu Trp Gly Arg Arg Ile Lys Asp Asp         515 520 525 Asp Thr Ser Ile Ala Tyr Ser Ser Arg Leu Met Ile Gln Gln Phe Glu     530 535 540 Arg Asp Gly Ser Asn Asn Thr Asp Trp Val Tyr Ser Asp His Ile Tyr 545 550 555 560 Phe Tyr Lys Leu Trp Lys Glu Val Ala Gly Lys Arg Ala Val Ser Thr                 565 570 575 Ser Thr Asp Leu Asp Ile Gln Ser Gln Val Phe Ile Asp Gly Lys Thr             580 585 590 Ala Tyr Val Val Leu Thr Ser Leu Glu Phe Asp Asp Arg Ala Ile Gln         595 600 605 Leu Asn Thr Leu Gly Val Asp Asn Asn Ser Leu Ser Ser Val Glu Ile     610 615 620 Lys Thr Leu Gly Tyr Ser Glu Gln Asn Thr Val Asp Tyr Gln Ile Thr 625 630 635 640 Asn Met Thr Glu Leu Pro Asp Asn Trp Val Leu Pro Ala Glu Ala Thr                 645 650 655 Gln Ile Ile Lys Leu Thr Tyr Ser Ser Asp Ile Asn Gln Lys Glu Ser             660 665 670 Ala Ser Thr Ile Lys Tyr Tyr Ala Ser Glu Tyr Leu Gln Ala Ile Gln         675 680 685 Ala Tyr Ser Pro Met Ser Phe Asn Ile Asp Asp Val Asn Val Gly Ser     690 695 700 Gln Gly Tyr Ala Val Leu Arg Met Gly Leu Gly Arg Asp His Gly Lys 705 710 715 720 Ser Leu Thr Pro Lys Val Thr Ile Asn Gly Val Thr Leu Thr Val Pro                 725 730 735 Ser Asp Phe Arg Gly Tyr Asp Gln Lys Gln Gly Lys Ser Lys Thr Gly             740 745 750 Arg Glu Asn Phe Phe Gly Val Ile Glu Ile Pro Val Pro Tyr Ser Ala         755 760 765 Leu Gln Ser Asp Asn Asn Val Glu Ile Glu Phe Ala Asp Asn Gly Gly     770 775 780 Tyr Val Ser Ser Ale Leu Gln Val Val Thr Ser Ser Lys Lys Ile 785 790 795 800 Gln Tyr Thr Leu Asn                 805 <210> 3 <211> 796 <212> PRT <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 3 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 Arg Gly Ser His Met Thr Lys Glu Pro Thr Ala Val Ile Asp Thr Ser              20 25 30 Pro Asp Arg Phe Thr Leu Asp Ala Ile Thr Gly Ile Ala Leu Asp Thr          35 40 45 Trp Val Thr Ser Lys Pro Phe Lys Val Ala Gly Ile Asn Ala Pro Thr      50 55 60 Ala Ile Lys Ile Thr Asp Gly Glu Tyr Ser Val Asn Gly Ser Ala Phe  65 70 75 80 Thr Asn Gln Ser Gly Thr Ile Lys Leu Gly Asp Ser Ile Ser Val Arg                  85 90 95 Ala Lys Ser Lys Ser Glu His Ala Ser Ser His Thr Ala Thr Leu Phe             100 105 110 Ile Gly Asp Lys Gln Ala Asp Phe Ile Ile Thr Thr Leu Ala Ala Pro         115 120 125 Thr Phe Asn Gly Thr Gln Val Asn Val His Leu Asp Thr Leu His Ser     130 135 140 Ile Asn Gly Ile Asp Ser Phe Glu Arg Glu Lys Tyr Ile Thr Ile His 145 150 155 160 Ser Ser Asn Val Glu Asn Asp Trp Gly Gln Asn Asp Ser His Ser Asn                 165 170 175 Asn Ala Ala Asn Ala Leu Gly Asp Asp Leu Val Phe Asp Phe Ile Glu             180 185 190 Gly His Asn Val Tyr Phe Gly Arg Glu Thr Gly Ser Leu Gly Trp Asn         195 200 205 Leu Arg Asn Val Arg Gln Asp Asp Ser Arg Pro Gly Tyr Val Asp Pro     210 215 220 Ala Ser Leu Ile Ser Arg Ala Gly Asp Ser Asn Trp Thr Tyr Asp Asn 225 230 235 240 Ser Gln Gln Ala Lys Phe Lys Asn Gly Arg Gln Leu Glu Asp Arg Met                 245 250 255 Ala Gly Met Ile Val Gly Gly Gln Gln His Pro Tyr Trp Pro Glu Gly             260 265 270 Thr Glu Ile Thr Pro Val Ala Lys Leu Ala Glu Pro Lys Trp Ser Phe         275 280 285 Ser Gln Thr Asp Thr Ala Asn Glu Pro Leu Gly Thr Ala Thr Gly Glu     290 295 300 Tyr Phe Ala Arg Tyr Leu Gln Asn Phe Tyr Arg Gln Ser Ser Glu Asp 305 310 315 320 Ala Gly Pro Lys Pro Lys Tyr Phe Glu Val Met Asn Glu Pro Leu                 325 330 335 Tyr Asp Leu Ser Thr Asp Arg Glu Gly Glu Thr Asn Tyr Val Glu Pro             340 345 350 Leu Lys Val Phe Glu Phe His Asn Thr Val Ala Lys Glu Ile Ala Lys         355 360 365 Ile Ser Asp Asn His Asp Ile Leu Val Gly Gly Tyr Thr Ile Ala Phe     370 375 380 Pro Asp Phe Asp Lys Asn Asn Phe Gln Arg Trp His Asp Arg Asp Lys 385 390 395 400 Leu Phe Ile Asp Thr Ser Gly Glu Tyr Met Asp Phe Tyr Ser Ile His                 405 410 415 Leu Tyr Asp Phe Pro Glu Phe Lys Asn Ser Glu Arg Tyr Arg Arg Gly             420 425 430 Ser Asn Leu Glu Ala Thr Leu Asp Met Leu Glu Gln Tyr Ser Gln Ile         435 440 445 Lys Leu Gly Glu Ile Lys Pro Ile Val Ile Ser Glu Val Gly Ser Ser     450 455 460 Thr His Ile Met Val Asn Gln Asp Trp Ser Pro Gln Arg Asp Ala Glu 465 470 475 480 Lys Val Arg Ser Ser Thre Ser Met Asn Thr Gln Phe Leu Glu Arg Pro                 485 490 495 Asp Thr Ile Ala Lys Val Ile Pro Phe Ile Pro Val Lys Ala Glu Trp             500 505 510 Gly Arg Arg Ile Lys Asp Asp Asp Thr Ser Ile Ala Tyr Ser Ser Arg         515 520 525 Leu Met Ile Gln Gln Phe Glu Arg Asp Gly Ser Asn Asn Thr Asp Trp     530 535 540 Val Tyr Ser Asp His Ile Tyr Phe Tyr Lys Leu Trp Lys Glu Val Ala 545 550 555 560 Gly Lys Arg Ala Val Ser Thr Ser Thr Asp Leu Asp Ile Gln Ser Gln                 565 570 575 Val Phe Ile Asp Gly Lys Thr Ala Tyr Val Val Leu Thr Ser Leu Glu             580 585 590 Phe Asp Asp Arg Ala Ile Gln Leu Asn Thr Leu Gly Val Asp Asn Asn         595 600 605 Ser Leu Ser Ser Val Glu Ile Lys Thr Leu Gly Tyr Ser Glu Gln Asn     610 615 620 Thr Val Asp Tyr Gln Ile Thr Asn Met Thr Glu Leu Pro Asp Asn Trp 625 630 635 640 Val Leu Pro Ala Glu Ala Thr Gln Ile Ile Lys Leu Thr Tyr Ser Ser                 645 650 655 Asp Ile Asn Gln Lys Glu Ser Ala Ser Thr Ile Lys Tyr Tyr Ala Ser             660 665 670 Glu Tyr Leu Gln Ala Ile Gln Ala Tyr Ser Pro Met Ser Phe Asn Ile         675 680 685 Asp Asp Val Asn Val Gly Ser Gln Gly Tyr Ala Val Leu Arg Met Gly     690 695 700 Leu Gly Arg Asp His Gly Lys Ser Leu Thr Pro Lys Val Thr Ile Asn 705 710 715 720 Gly Val Thr Leu Thr Val Pro Ser Asp Phe Arg Gly Tyr Asp Gln Lys                 725 730 735 Gln Gly Lys Ser Lys Thr Gly Arg Glu Asn Phe Phe Gly Val Ile Glu             740 745 750 Ile Pro Val Pro Tyr Ser Ala Leu Gln Ser Asp Asn Asn Val Gln Ile         755 760 765 Glu Phe Ala Asp Asn Gly Gly Tyr Val Ser Ser Ala Leu Gln Val     770 775 780 Val Thr Ser Ser Lys Lys Ile Gln Tyr Thr Leu Asn 785 790 795 <210> 4 <211> 2328 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 4 actaaagaac caactgccgt tatcgatacc agccctgacc gctttaccct tgacgcaata 60 acaggcattg ctttagatac ttgggtaaca agcaaaccct ttaaagtagc aggtattaat 120 gcgccgaccg caataaaaat aacggatgga gaatactctg ttaatggctc agcttttact 180 aatcaaagcg gcactataaa gctgggcgat agcatatctg tcagagctaa aagcaaatct 240 gagcacgcta gttcccatac cgccacttta ttcattggcg ataaacaagc agactttatt 300 atcaccacct tagcggcccc tacgtttaat ggcacccagg tcaatgtgca tttagatacc 360 ttgcacagca taaatggcat tgatagtttt gagcgcgaaa aatacatcac aatacattca 420 agcaacgtcg aaaatgactg gggacaaaac gatagccata gtaacaatgc tgccaatgca 480 ttaggtgatg acttagtgtt tgattttatt gaaggccata acgtttattt tggacgtgaa 540 actggtagct tgggctggaa tttacgcaat gttcgccaag atgatagccg acccggttat 600 gttgaccctg ccagtctaat aagccgcgcg ggcgattcta actggacata cgacaacagc 660 cagcaagcta aatttaaaaa tggccgtcaa cttgaagatc gcatggccgg aatgatagtt 720 ggtggccaac agcaccctta ttggccagaa ggcacagaga tcacgccagt ggcaaaatta 780 gctgagccaa aatggtcatt ttcacagaca gatactgcta atgaaccatt aggaactgcc 840 accggagaat attttgctcg atatttgcaa aacttttacc gtcaatcatc agaagatgct 900 gggcccccta aacctaaata ctttgaagtg atgaacgaac ctttatacga tttaagtaca 960 gacagagaag gtgaaacaaa ttatgtggag ccgctaaaag tctttgagtt tcataatact 1020 gtggccaaag agatagctaa aattagtgac aaccatgata ttttagtggg aggttatacg 1080 atagcttttc cagattttga taaaaacaac tttcagcgct ggcatgacag agacaaattg 1140 tttattgata catccggcga atacatggat ttttattcaa tccatttgta cgattttcct 1200 ggtttaaaa actcagagcg ttatcgccgt ggcagtaatt tagaagccac tttagatatg 1260 ttagagcaat atagccaaat aaaacttggg gaaattaaac caattgtaat ttccgaagtg 1320 ggctcatcga ctcatatcat ggttaaccaa gattggagcc cgcaaaggga tgccgaaaag 1380 gtacgctcaa ttaccagcat gaatactcag tttttagagc gtcccgatac gatagctaaa 1440 gtgatccctt ttattcctgt gaaagcagaa tggggtcgtc gaataaaaga tgatgatacc 1500 agcatcgcct attcgagccg tttgatgatc caacagtttg agcgtgatgg cagcaataat 1560 acagactggg tttattctga tcatatttat ttttacaaac tatggaaaga ggtagctggt 1620 aaacgcgccg ttagcacaag cacagactta gatattcaaa gccaagtatt tattgatggt 1680 aaaacggctt atgtggtgtt aaccagctta gagtttgatg atagagcaat ccagttgaat 1740 actttaggcg ttgataataa ttcattaagt tcggttgaaa tcaaaacgct tggttatagt 1800 gagcaaaata cagttgatta tcaaatcact aatatgactg agttgcctga taattgggtt 1860 ttacctgccg aagccactca aattatcaaa ctcacttata gcagtgatat caatcaaaaa 1920 gaatcagcga gtacaattaa atattatgct agtgagtact tacaggcaat acaagcctat 1980 agtccaatga gttttaacat cgatgatgtg aatgttggca gtcaaggata tgctgtgcta 2040 agaatgggtt tagggcgcga tcatggtaaa tccttaacac caaaagtaac aattaatggt 2100 gtcacgctaa ccgtacccag tgattttaga ggttatgatc aaaaacaagg gaaatcaaaa 2160 actggccgcg aaaacttttt tggtgtgata gagatccctg tgccttactc agcactgcaa 2220 agcgataata atgttcagat tgagtttgct gacaacggcg gttatgtcag tagtatggcg 2280 ctgcaagtgg tcactagcag taaaaagatt caatatacac ttaattaa 2328 <210> 5 <211> 2418 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 5 atgaataaag cctttgtgag caataaacta agtttagcga tttgtgtaag ccttttttta 60 acggcctgtg gtggtggcgg ttcgacagaa actaaagaac caactgccgt tatcgatacc 120 agccctgacc gctttaccct tgacgcaata acaggcattg ctttagatac ttgggtaaca 180 agcaaaccct ttaaagtagc aggtattaat gcgccgaccg caataaaaat aacggatgga 240 gaatactctg ttaatggctc agcttttact aatcaaagcg gcactataaa gctgggcgat 300 agcatatctg tcagagctaa aagcaaatct gagcacgcta gttcccatac cgccacttta 360 ttcattggcg ataaacaagc agactttatt atcaccacct tagcggcccc tacgtttaat 420 ggcacccagg tcaatgtgca tttagatacc ttgcacagca taaatggcat tgatagtttt 480 gagcgcgaaa aatacatcac aatacattca agcaacgtcg aaaatgactg gggacaaaac 540 gatagccata gtaacaatgc tgccaatgca ttaggtgatg acttagtgtt tgattttatt 600 gaaggccata acgtttattt tggacgtgaa actggtagct tgggctggaa tttacgcaat 660 gttcgccaag atgatagccg acccggttat gttgaccctg ccagtctaat aagccgcgcg 720 ggcgattcta actggacata cgacaacagc cagcaagcta aatttaaaaa tggccgtcaa 780 cttgaagatc gcatggccgg aatgatagtt ggtggccaac agcaccctta ttggccagaa 840 ggcacagaga tcacgccagt ggcaaaatta gctgagccaa aatggtcatt ttcacagaca 900 gatactgcta atgaaccatt aggaactgcc accggagaat attttgctcg atatttgcaa 960 aacttttacc gtcaatcatc agaagatgct gggcccccta aacctaaata ctttgaagtg 1020 atgaacgaac ctttatacga tttaagtaca gacagagaag gtgaaacaaa ttatgtggag 1080 ccgctaaaag tctttgagtt tcataatact gtggccaaag agatagctaa aattagtgac 1140 aaccatgata ttttagtggg aggttatacg atagcttttc cagattttga taaaaacaac 1200 tttcagcgct ggcatgacag agacaaattg tttattgata catccggcga atacatggat 1260 ttttattcaa tccatttgta cgattttcct gagtttaaaa actcagagcg ttatcgccgt 1320 ggcagtaatt tagaagccac tttagatatg ttagagcaat atagccaaat aaaacttggg 1380 gaaattaaac caattgtaat ttccgaagtg ggctcatcga ctcatatcat ggttaaccaa 1440 gattggagcc cgcaaaggga tgccgaaaag gtacgctcaa ttaccagcat gaatactcag 1500 tttttagagc gtcccgatac gatagctaaa gtgatccctt ttattcctgt gaaagcagaa 1560 tggggtcgtc gaataaaaga tgatgatacc agcatcgcct attcgagccg tttgatgatc 1620 caacagtttg agcgtgatgg cagcaataat acagactggg tttattctga tcatatttat 1680 ttttacaaac tatggaaaga ggtagctggt aaacgcgccg ttagcacaag cacagactta 1740 gatattcaaa gccaagtatt tattgatggt aaaacggctt atgtggtgtt aaccagctta 1800 gagtttgatg atagagcaat ccagttgaat actttaggcg ttgataataa ttcattaagt 1860 tcggttgaaa tcaaaacgct tggttatagt gagcaaaata cagttgatta tcaaatcact 1920 aatatgactg agttgcctga taattgggtt ttacctgccg aagccactca aattatcaaa 1980 ctcacttata gcagtgatat caatcaaaaa gaatcagcga gtacaattaa atattatgct 2040 agtgagtact tacaggcaat acaagcctat agtccaatga gttttaacat cgatgatgtg 2100 aatgttggca gtcaaggata tgctgtgcta agaatgggtt tagggcgcga tcatggtaaa 2160 tccttaacac caaaagtaac aattaatggt gtcacgctaa ccgtacccag tgattttaga 2220 ggttatgatc aaaaacaagg gaaatcaaaa actggccgcg aaaacttttt tggtgtgata 2280 gagatccctg tgccttactc agcactgcaa agcgataata atgttcagat tgagtttgct 2340 gacaacggcg gttatgtcag tagtatggcg ctgcaagtgg tcactagcag taaaaagatt 2400 caatatacac ttaattaa 2418 <210> 6 <211> 2391 <212> DNA <213> Unknown <220> <223> Gayadomonas joobiniege G7 <400> 6 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgactaaag aaccaactgc cgttatcgat accagccctg accgctttac ccttgacgca 120 ataacaggca ttgctttaga tacttgggta acaagcaaac cctttaaagt agcaggtatt 180 aatgcgccga ccgcaataaa aataacggat ggagaatact ctgttaatgg ctcagctttt 240 actaatcaaa gcggcactat aaagctgggc gatagcatat ctgtcagagc taaaagcaaa 300 tctgagcacg ctagttccca taccgccact ttattcattg gcgataaaca agcagacttt 360 attatcacca ccttagcggc ccctacgttt aatggcaccc aggtcaatgt gcatttagat 420 accttgcaca gcataaatgg cattgatagt tttgagcgcg aaaaatacat cacaatacat 480 tcaagcaacg tcgaaaatga ctggggacaa aacgatagcc atagtaacaa tgctgccaat 540 gcattaggtg atgacttagt gtttgatttt attgaaggcc ataacgttta ttttggacgt 600 gaaactggta gcttgggctg gaatttacgc aatgttcgcc aagatgatag ccgacccggt 660 tatgttgacc ctgccagtct aataagccgc gcgggcgatt ctaactggac atacgacaac 720 agccagcaag ctaaatttaa aaatggccgt caacttgaag atcgcatggc cggaatgata 780 gttggtggcc aacagcaccc ttattggcca gaaggcacag agatcacgcc agtggcaaaa 840 ttagctgagc caaaatggtc attttcacag acagatactg ctaatgaacc attaggaact 900 gccaccggag aatattttgc tcgatatttg caaaactttt accgtcaatc atcagaagat 960 gctgggcccc ctaaacctaa atactttgaa gtgatgaacg aacctttata cgatttaagt 1020 acagacagag aagattagaac aaattatgtg gagccgctaa aagtctttga gtttcataat 1080 actgtggcca aagagatagc taaaattagt gacaaccatg atattttagt gggaggttat 1140 acgatagctt ttccagattt tgataaaaac aactttcagc gctggcatga cagagacaaa 1200 ttgtttattg atacatccgg cgaatacatg gatttttatt caatccattt gtacgatttt 1260 cctgagttta aaaactcaga gcgttatcgc cgtggcagta atttagaagc cactttagat 1320 atgatagagc aatatagcca aataaaactt ggggaaatta aaccaattgt aatttccgaa 1380 gtgggctcat cgactcatat catggttaac caagattgga gcccgcaaag ggatgccgaa 1440 aaggtacgct caattaccag catgaatact cagtttttag agcgtcccga tacgatagct 1500 aaagtgatcc cttttattcc tgtgaaagca gaatggggtc gtcgaataaa agatgatgat 1560 accagcatcg cctattcgag ccgtttgatg atccaacagt ttgagcgtga tggcagcaat 1620 aatacagact gggtttattc tgatcatatt tatttttaca aactatggaa agaggtagct 1680 ggtaaacgcg ccgttagcac aagcacagac ttagatattc aaagccaagt atttattgat 1740 ggtaaaacgg cttatgtggt gttaaccagc ttagagtttg atgatagagc aatccagttg 1800 aatactttag gcgttgataa taattcatta agttcggttg aaatcaaaac gcttggttat 1860 agtgagcaaa atacagttga ttatcaaatc actaatatga ctgagttgcc tgataattgg 1920 gttttacctg ccgaagccac tcaaattatc aaactcactt atagcagtga tatcaatcaa 1980 aaagaatcag cgagtacaat taaatattat gctagtgagt acttacaggc aatacaagcc 2040 tatagtccaa tgagttttaa catcgatgat gtgaatgttg gcagtcaagg atatgctgtg 2100 ctaagaatgg gtttagggcg cgatcatggt aaatccttaa caccaaaagt aacaattaat 2160 ggtgtcacgc taaccgtacc cagtgatttt agaggttatg atcaaaaaca agggaaatca 2220 aaaactggcc gcgaaaactt ttttggtgtg atagagatcc ctgtgcctta ctcagcactg 2280 caaagcgata ataatgttca gattgagttt gctgacaacg gcggttatgt cagtagtatg 2340 gcgctgcaag tggtcactag cagtaaaaag attcaatata cacttaatta a 2391

Claims (14)

삭제delete 서열번호 1의 아미노산 서열을 포함하는 베타-아가레이즈를 유효성분으로 함유하는 네오아가로헥사오스(neoagarohexaose) 생산용 효소조성물.An enzyme composition for producing neoagarohexaose containing beta-agarase containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 서열번호 1의 아미노산 서열을 포함하는 베타-아가레이즈를 유효성분으로 함유하는 아가로오스(agarose) 분해용 효소조성물.An enzyme composition for agarose degradation comprising a beta-agarase containing the amino acid sequence of SEQ ID NO: 1 as an active ingredient. 서열번호 1의 아미노산 서열을 포함하는 베타-아가레이즈를 코딩하는 베타-아가레이즈 유전자.A beta-agarase gene encoding a beta-agarase comprising the amino acid sequence of SEQ ID NO: 1. 제 4항에 있어서,
상기 유전자는 서열번호 4의 염기서열을 포함하는 것을 특징으로 하는 베타-아가레이즈 유전자.5. The method of claim 4,
Wherein the gene comprises the nucleotide sequence of SEQ ID NO: 4. 제 4항의 유전자를 함유하는 베타-아가레이즈 생산용 재조합 벡터.A recombinant vector for producing beta-agarase containing the gene of claim 4. 제 6항의 재조합 벡터로 형질전환된 베타-아가레이즈 생산용 형질전환체.A transformant for producing beta-agarase transformed with the recombinant vector of claim 6. 제 7항의 형질전환체를 배양하고 상기 베타-아가레이즈 유전자를 과발현시키는 것을 특징으로 하는 베타-아가레이즈 대량생산방법.A method for mass production of a beta-agarase comprising culturing the transformant of claim 7 and overexpressing the beta-agarase gene. 서열번호 1의 아미노산 서열을 포함하는 베타-아가레이즈를 아가로오스와 효소반응시키는 것을 특징으로 하는 네오아가로헥사오스 생산방법.Characterized in that a beta-agarase containing the amino acid sequence of SEQ ID NO: 1 is subjected to an enzymatic reaction with agarose to produce neoagaroohexaose. 제 9항에 있어서,
상기 효소반응이 pH 4 내지 6에서 이루어지는 것을 특징으로 하는 네오아가로헥사오스 생산방법.10. The method of claim 9,
Wherein the enzyme reaction is carried out at a pH of from 4 to 6. &lt; RTI ID = 0.0 &gt; 5. &lt; / RTI &gt; 제 9항에 있어서,
상기 효소반응이 10 내지 40℃에서 이루어지는 것을 특징으로 하는 네오아가로헥사오스 생산방법.10. The method of claim 9,
Wherein the enzyme reaction is carried out at 10 to 40 占 폚. 서열번호 1의 아미노산 서열을 포함하는 베타-아가레이즈를 아가로오스와 효소반응시키는 것을 특징으로 하는 아가로오스 분해방법.Characterized in that a beta-agarase containing the amino acid sequence of SEQ ID NO: 1 is subjected to an enzymatic reaction with agarose. 제 12항에 있어서,
상기 효소반응이 pH 4 내지 6에서 이루어지는 것을 특징으로 하는 아가로오스 분해방법.13. The method of claim 12,
Wherein the enzymatic reaction is carried out at a pH of 4 to 6. 제 12항에 있어서,
상기 효소반응이 10 내지 40℃에서 이루어지는 것을 특징으로 하는 아가로오스 분해방법.
13. The method of claim 12,
Wherein the enzyme reaction is carried out at 10 to 40 占 폚.
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Title
Appl Biochem Biotechnol [DOI 10.1007/s12010-016-2262-x]
J. Microbiol. Biotechnol. (2018), VOl. 28(2), p.284-292 (published online November 25, 2017)
Lee Y.R. et al, J. Microbiol. Biotechnol. 28(2):pp.284~292 (2017.11.25.)*
NCBI Reference Sequence WP_017446675.1 (2013. 6.28)*
NCBI Reference Sequence: WP_017446675.1

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