Purification of Cold-adapted Protease from Janthinobacterium sp.

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June 2018 | Vol. 46 | No. 2 Microbiol. Biotechnol. Lett. (2018), 46(2), 111–113

http://dx.doi.org/10.4014/mbl.1802.02016 pISSN 1598-642X eISSN 2234-7305

Microbiology and Biotechnology Letters

Janthinobacterium sp. 유래 저온 활성 프로테아제 정제

김현도, 최종일*

전남대학교생물공학과, 바이오에너지및바이오소재협동과정

Received: March 6, 2018 / Revised: March 8, 2018 / Accepted: March 9, 2018

일반적으로극지와같은특수한환경에서생장하는생물 종들은생물산업에많은기회를제공해준다^.최근에는이러 한저온에서적응하며생존하는종들을생물산업의응용을

위한연구모델로활용하고있다^{[1, 2].}극지와같은저온환

경에서생장하는미생물에서분리된효소는저온및중온에 서높은촉매활성을나타내기때문에^,반응조건이무기촉 매나다른효소에비하여용이하며^,또한적은온도상승에 의해쉽게불활성화될수있기때문에산업적으로응용가 치가높고그에대한연구가증가하고있다^[3−^5].

세계효소시장의약 60%를차지하는프로테아제는세제 산업^,가죽가공^,식품산업^,의약품및생물학적환경정화등 수많은분야에서잠재적인이점을갖는효소이다^[6].특히^,저 온성프로테아제는세제및식품가공과같이저온에서반응 이수행되어야하는산업분야에서시간과에너지소비를줄 일수있으므로효율적으로공정을수행할수있게한다^[7−^9].

지금까지여러종류의저온미생물로부터다양한프로테 아제가보고되었다^[7−^{9]. Yang}등은 Halobacillus에서크로 마토그래피를이용하여저온활성프로테아제를정제하였으 며[9], Yersinia ruckeri에서암모니움설페이트침전과이온

크로마토그래피를이용하여프로테아제가정제되었다^[10].

Davail 등은 phenyl-Sepharose CL-4B colum을이용하여저 온성^Bacillus에서^alkaline프로테아제를정제하였다^[11].

하지만^,이러한저온활성효소의분리와정제는효소의특 성에따라조건이다양하고^,온도에따른활성변화가쉽게 야기될수있는경우가많기때문에다양한효소에대한정 제방법연구가필요한실정이다^.따라서^,본논문에서는이 전연구에서선별된극지미생물들중프로테아제의활성이 높은 Janthinobacterium sp. PAMC 25641로부터 분비된 프로테아제의정제연구를수행하였다[12].

본 실험에서 사용된 Janthinobacterium sp. PAMC 25641은 극지연구소(Korea Polar Research Institute, Korea)로부터 분양받았다^.이전 연구에따르면 Janthino- bacterium sp. 다른국지유래균주에비하여성장이우수하 고^,세포외로분비된프로타아제는저온에서높은활성을나 타내는것으로확인하였다^[12].

Janthinobacterium sp.는 250 ml erlenmeyer flasks에 50 ml NB 배지(Nutrient broth, Becton, Dickinson and Company, USA)를이용하여¹⁵℃에서²⁴시간전배양하였 고본배양은같은조건에서전배양액의^2%(v/v)를접종하여 72시간동안배양하였다^.미생물의 성장은 ELISA Reader (Molecular Devices, VersaMax^TM and SpetraMax^® 340PC³⁸⁴, USA)를사용하여^{600 nm}에서흡광도를측정하였다^. Purification of Cold-adapted Protease from Janthinobacterium sp.

Hyun-do Kim and Jong-il Choi*

Department of Biotechnology and Bioengineering, Interdisciplinary Program for Bioenergy & Biomaterials, Chonnam National University, Gwangju 61186, Republic of Korea

In this study, purification of cold-adapted protease from Janthinobacterium sp. was investigated. First, using gradient precipitation, protease was confirmed to be deposited in the 30−80% range of ammonium sulfate. Next, DEAE-Sepharose column was used for the binding of the protease under various conditions.

The optimal binding condition was found to be pH 8.5 and flow rate of 30 ml/h. Under the optimal condition, the protease was purified with 29% recovery yield. This result can be useful for the purification of other cold-adapted protein.

Keywords: Purification, protease, Janthinobacterium

*Corresponding author

Tel: +82-62-530-1846, Fax: +82-62-530-1949 E-mail: [email protected]

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112 Kim and Choi

http://dx.doi.org/10.4014/mbl.1802.02016

단백질의농도는 Bradford assay 방법을이용하여측정하 였다^[13].

Janthinobacterium sp. 유래 프로테아제 활성 측정은 50 mM sodium phosphate buffer (pH 7.2) 200 µ^l에기질 인 10 mM AAPF (N-succinyl-ALA-ALA-PRO-PHE-P- nitroanilide, Sigma-Aldrich, USA) 10µ^l을넣고¹⁰⁰µ^l의 효소혼합액과⁶⁹⁰µ^l의증류수를혼합하여²⁵^o^C에서¹⁰분 간 반응시킨 후⁶⁰℃에서²⁰분간 불활성화 하여^{, ELISA} reader를사용하여^{410 nm}에서흡광도를측정하였다^{. 1 unit} 의프로테아제활성은 extinction coefficient = 8,800으로하 여다음식에의하여계산하였다^[12].

Unit (mmol/min)/l =

Janthinobacterium sp.로부터세포외로분비된프로테아 제를분리하기위하여먼저 gradient precipitation을수행하 였다^.발효후얻어진여액에암모늄설페이트를⁰−^100%농 도범위로처리한후⁴℃, 22,000 ×g으로³⁰분간원심분리 하였다^.원심분리후얻은^pellet을 20 mM Tris-HCl buffer (pH 8.5)로녹인후같은용액으로투석을진행하였다^.투석 용액은적어도²번교체하였고^,침전을위해사용된암모늄 설페이트농도에대한상대적인프로테아제의활성을투석 후측정하였다^.

이러한방법을통해서최적의암모늄설페이트농도를설 정하였고^,이후정제단계의최적화를진행하였다^.발효후 얻어진여액에암모늄설페이트를^70%의농도로처리한후 4℃, 22,000 ×g으로³⁰분간원심분리하였다^.원심분리후 얻은^pellet을 20 mM Tris-HCl buffer (pH 8.5)로녹인 후 같은용액으로투석을진행하였다^.투석용액은적어도²번 교체하였다^.크로마토그래피를이용한단백질정제의최적 화를위하여다양한조건의^{pH (6.5}−^8.5)와유속^(1.5−^{3 ml/}

min)에서정제를수행하고분석하였다^.투석후원심분리하 여분리된상층액을 DEAE-Sepharose column (1.5 × 5 cm, Sigma-Aldrich)에가하였고⁰−^{0.5 M}농도의^NaCl을^{30 ml/h}의

유속으로^{10 ml}씩용출하였고각각의분획의효소활성을

측정하였다^.

Janthinobacterium sp.로부터생산된프로테아제의최적 침적범위를확인하기위하여³⁰−^80%범위의암모늄설페 이트를이용하여 gradient precipitation을수행하였다^.그결 과^,프로테아제의최적침전범위는⁶⁰−^70%로확인되었다 (Fig. 1). 30% 이하의암모늄암모늄설페이트농도에서는프 로테아제는침전이일어나지않았으며^{, 40%}이상에서프로 테아제침전이확인되었다^.침전된프로테아제의활성은암 (net OD410) × (volume of reaction mixture, L) × 10⁶)

ε × (reaction time, min) × (volume of enzyme solution, L)

Fig. 1. Protease activity after gradient precipitation of cell broth of Janthinobacterium sp..

Fig. 2. Protease activity of fractions after elution. The frac- tions were eluted with 0−0.5 M NaCl gradient at a flow rate of 30 ml/h, with 10 mM of the fraction volume.

Fig. 3. SDS-PAGE analysis of fractions after elution.

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모늄설페이트의농도가증가할수록증가하여^70%에서가 장높은값을가졌다^.

따라서^,배양액으로부터프로테아제의일차적인정제를위 한침전조건을^70%의암모늄설페이트로설정하고^,침전되 어얻어진프로테아제의고순도정제를위한크로마토그래 프를수행하였다^.침전으로부터얻어진프로테아제용액을

DEAE-Sepharose 컬럼의단백질결합을최적화하기위하

여실험을수행한결과^{, pH 7.5}−^8.0에서는^{pH 8.5}보다낮 은단백질결합을나타내었다^{. pH 8.5}에서유속은단백질결 합정도에영향을 미치지않았다(data not shown). 따라서 DEAE-Sepharose의 로딩 조건을^{pH 8.5,}유속^{30 ml/h}로 설정하였다^.

DEAE-Sepharose에결합된프로테아제의용출을통하여

정제된효소의활성은⁷⁰−^{120 mM}농도의^NaCl을이용하 여용출한분획⁸−¹³사이에서확인하였다^{(Fig. 2).}프로테 아제의활성이가장높은분획은분획¹⁰이었으며^,분획⁸에 서¹²까지의용출액을모아서측정한효소의수율은^29%로 확인하였다^. 분획정제된 프로테아제의 분자량은 대략 110 kDa으로확인되어졌다^{(Fig. 3).}

Janthinobacterium sp.의배양상등액으로부터프로테아 제를분리하기위한단계별활성^,총단백질양및정제수율 을^{Table 1}에나타내었다^.

극지와같은저온환경에서생장하는미생물이분비하는 프로테아제는여러산업분야에서매우유용하게사용될수 있다^.따라서본연구의결과는산업적이용가치가높은다 양한저온활성효소의정제와그활용에효과적으로이용될 수있을것이다^.

Acknowledgement

This research was supported by Golden Seed Project, Ministry of Oceans and Fisheries (213008-05-2-SB910).

Conflict of Interest

The authors have no financial conflicts of interest to declare.

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Table 1. Protease activity and recovery yield during purification steps obtained from the culture broth of Janthinobacterium sp..

Step Total volume

(ml)

Activity (U/l)

Total activity recovered (U)

Protein (mg)

Specific activity (U/mg protein)

Recovery yield (%)

Cell-free culture supernatant 1000 40 40,000 1,290 31 100

Ammonium sulfate precipitation 40 980 39,200 243 161 98

DEAE-Sepharose fraction (8-12) 60 195 11,700 20 584 29