KR101235289B1 - A Novel yeast strain of Saccharomyces servazzii KU 244 producing β-glucosidase isolated from Kimchi - Google Patents

A Novel yeast strain of Saccharomyces servazzii KU 244 producing β-glucosidase isolated from Kimchi Download PDF

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KR101235289B1
KR101235289B1 KR1020110007063A KR20110007063A KR101235289B1 KR 101235289 B1 KR101235289 B1 KR 101235289B1 KR 1020110007063 A KR1020110007063 A KR 1020110007063A KR 20110007063 A KR20110007063 A KR 20110007063A KR 101235289 B1 KR101235289 B1 KR 101235289B1
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백현동
조미나
한은진
이나경
장경훈
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건국대학교 산학협력단
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Abstract

본 발명은 우리나라 김치에서 ß-glucosidase를 생산하는 균주 KU244를 최초로 분리하고, Saccharomyces servazzii로 동정한 후 Saccharomyces servazzii KU 244로 명명하였으며, 이 균주는 배양 24시간때까지 급격히 ß-glucosidase 효소활성이 증가하며, 그 이후로는 서서히 증가하는 것을 확인하는 특징이 있고, 반응온도 50℃에서 상등액 1 mL의 효소활성은 13.04 μM/ min·mL로서 최대값을 나타내었으며 반응 최적온도는 50℃로 확인되어 생물의료산업 상 매우 유용한 발명인 것이다.The present invention is to remove the strain KU244 to produce ß-glucosidase from Korean kimchi first, and then identified as Saccharomyces servazzii was named Saccharomyces servazzii KU 244, the strain increases sharply ß-glucosidase enzyme activity until the cultivation for 24 hours, and After that, there was a characteristic of gradually increasing, and the enzyme activity of the supernatant 1 mL at the reaction temperature of 50 ℃ showed a maximum value of 13.04 μM / min · mL and the optimum temperature of the reaction was confirmed as 50 ℃ It is an industrially useful invention.

Description

김치유래의 베타 글루코시다아제 생산 신규한 효모 사카로마이세스 서바지 ＫＵ 244 균주{A Novel yeast strain of Saccharomyces servazzii KU 244 producing β-glucosidase isolated from Kimchi}A Novel yeast strain of Saccharomyces servazzii KU 244 producing β-glucosidase isolated from Kimchi

본 발명은 화장품, 와인, 생리활성물질, 세척제 및 직물산업 등 다양한 이용성이 있는 ß-glucosidase를 생산하는 김치유래의 신규한 효모균주와 그것에서부터 분리정제한 상기 효소의 특성에 관한 것이다.
The present invention relates to a novel yeast strain derived from Kimchi that produces ß-glucosidase with various uses, such as cosmetics, wine, bioactive substances, cleaning agents and textile industries, and the characteristics of the enzyme isolated and purified therefrom.

김치는 한국의 대표적인 전통 채소 발효식품으로 유기산, 식이섬유, 생리활성물질, 다량의 영양성분 및 인체에 유용한 미생물을 함유하고 있다. 이러한 이유로 김치는 해외에서도 점차 건강식품으로 인식되고 있으며, 김치의 기능성과 효능에 관한 연구(김창곤 외, 2005)와 김치유래 미생물(심종현, 2005; 장동훈 외, 2004; 이연희 외, 2004)에 관한 연구가 활발하게 진행되고 있다. 김치는 숙성 정도에 따라 미생물의 조성이 변하고, 발효 산물도 달라져 김치의 맛이 달라지게 된다. 이 등(1992)은 김치의 발효 초기에 Lactobacillus brevis와 L. sakei가, 과숙기에는 L. plantarum이, 적숙기에는 Leuconostoc sp.과 Lactobacillus sp.가 주로 분리되었으며, 또한 최(1978)에 의해 김치에서 Saccharomyces cerevisiae를 비롯하여 총 8속 13종의 효모가 분리되었다. Kimchi is a representative traditional Korean fermented food containing organic acid, dietary fiber, physiologically active substance, large amounts of nutrients and microorganisms useful for human body. For this reason, kimchi is increasingly recognized as a health food abroad, and studies on the function and efficacy of kimchi (Kim Chang-gon et al., 2005) and Kimchi-derived microorganisms (Shim Jong-hyun, 2005; Jang Dong-hoon et al., 2004; Lee Yeon-hee et al., 2004) Is actively underway. Kimchi changes the composition of the microorganisms depending on the degree of ripening, fermentation products are also changed, the taste of kimchi will change. Lee et al. (1992) found that Lactobacillus was the first to ferment kimchi. brevis and L. sakei , L. plantarum in ripening, and Leuconostoc in ripening sp. and Lactobacillus sp. was isolated mainly, and Choi (1978) isolated 13 yeast species from 8 genera including Saccharomyces cerevisiae .

한편, ß-glucosidase는 셀룰로오스나 ß-1,4 결합을 하고 있는 기질에서 포도당을 유리시키는 효소로서 그 생리적 기능은 식물에서 호르몬이나 향기성분 생산을 활성화 하는데 관여하거나, 식물 병원균에 대한 저항성 기작에 관여하는 것으로 알려져 있다(Estruch et al., 1991; Gunata et al., 1985). ß-Glucosidase는 넓은 기질 특이성을 이용하여, 산업적으로는 식품, 화학, 세척제 및 직물산업에 이용되고 있으며, ß-glucosidase를 함유한 미백제에 관한 연구가 보고된 바 있다.(박덕훈 외, 2003) 또, 와인 생산공정에서도 유용하게 사용되고 있다(김재영 외, 2009). 그런데, 이같은 glucosidase를 생산하는 미생물로는 Weissella cibaria(홍성욱 외, 2009), Lactobacillus sp., Leuconostoc sp.(장미희 외, 2010), Penicillium citrinum(Ng et al., 2010) 등이 보고되고 있으며, 최근에는 ß-glucosidase를 이용하여 이소플라본(홍성욱 외, 2009), 진세노사이드(유완식 외, 2009; 허율, 2002; Cheng et al., 2008)와 같은 배당체를 가수분해하여 생리활성물질을 얻고자 하는 연구들이 시도되고 있다. On the other hand, ß-glucosidase is an enzyme that releases glucose from cellulose or ß-1,4 binding substrate, and its physiological function is involved in activating the production of hormones or odor components in plants or in the mechanism of resistance to plant pathogens. (Estruch et al., 1991; Gunata et al., 1985). ß-Glucosidase is widely used in the food, chemical, detergent and textile industries for its broad substrate specificity, and studies have been reported on whitening agents containing ß-glucosidase (Park Deok-Hun et al., 2003). It is also usefully used in the wine production process (Kim Jae-young et al., 2009). By the way, microorganisms producing such glucosidase are Weissella. cibaria (Hong Sung-uk et al., 2009), Lactobacillus sp. , Leuconostoc sp. (Chang Mi-hee et al., 2010), Penicillium citrinum (Ng et al., 2010) has been reported, and recently, ß-glucosidase isoflavones (Hong Sung-wook et al., 2009), ginsenosides (Yu et al., 2009; Hur Yul, 2002; Cheng et al., Researches are attempting to obtain bioactive substances by hydrolyzing glycosides such as 2008).

따라서 본 발명은 다양한 분야에서 이용성이 높은 ß-glucosidase를 생산하는 신규한 효모 균주를 전통발효 김치로부터 최초로 분리하고, 거기로부터 생산된 ß-glucosidase의 이용 가능성을 제공하는데 그 목적이 있다.
Therefore, an object of the present invention is to first isolate a new yeast strain producing ß-glucosidase which is highly available in various fields from traditional fermented kimchi, and to provide the availability of ß-glucosidase produced therefrom.

본 발명의 상기 목적은 우리나라의 전통발효 김치에서 분리한 효모균주를 esculin 배지에 접종, 배양하여 ß-glucosidase 생산 균주를 선별하는 단계와 선별된 상기 균주를 동정하고 명명하는 단계와 상기 신규한 효모균주를 회분배양하면서 상기 ß-glucosidase 의 활성 및 온도특성을 확인하는 단계를 통하여 달성하였다.The object of the present invention is to inoculate and culture the yeast strain isolated from the traditional fermented kimchi of Korea in esculin medium to select the ß-glucosidase production strain and to identify and name the selected strain and the novel yeast strain While batch culture was achieved through the step of confirming the activity and temperature characteristics of the ß-glucosidase.

본 발명의 구체적인 내용은 실시에서 실험예에 의하여 명료히 하였다.
Specific details of the present invention were made clear by the experimental example in the implementation.

본 발명은 우리나라 전통발효 김치유래의 ß-glucosidase 생산을 신규한 효모균주와 상기 다양한 용도의 ß-glucosidase를 최초로 제공하는 효과가 있다.
The present invention has the effect of providing the ß-glucosidase of the traditional fermentation Kimchi-derived Korean yeast and the novel ß-glucosidase for the various uses for the first time.

도 1은 본 발명 김치유래의 신규한 효모균주 KU 244의 갈색환을 형성한 Colony를 보인 사진도이다.
도 2는 본 발명 신규한 효모균주 KU 244의 phylogenetic tree를 보인 그림이다.
도 3은 본 발명 신규한 효모균주 KU 244의 세포성장과 ß-glucosidase의 활성을 보인 그래프이다.
도 4는 본 발명 신규한 효모균주 KU 244로부터 분리한 ß-glucosidase 효모의 반응최적도 특성을 보인 그림이다.1 is a photograph showing a colony forming a brown ring of a novel yeast strain KU 244 derived from the kimchi of the present invention.
Figure 2 is a diagram showing the phylogenetic tree of the novel yeast strain KU 244 of the present invention.
3 is a graph showing the cell growth and ß-glucosidase activity of the novel yeast strain KU 244 of the present invention.
Figure 4 is a diagram showing the reaction optimum characteristics of ß-glucosidase yeast isolated from the novel yeast strain KU 244 of the present invention.

균주의 분리Isolation of strain

ß-Glucosidase를 생산하는 효모균주는 하기 esculin 배지를 이용하여 esculline 방법(Hernandez et al., 2003)을 사용하여 선별하였다. 김치에서 분리한 71종의 균주를 esculin 배지에 3 μL를 접종하여 배양하고, 갈색 환을 나타내는 균주를 선별하여 본 발명의 공시재료로 사용하였다. Yeast strains producing ß-Glucosidase were selected using the esculline method (Hernandez et al., 2003) using the following esculin medium. 71 strains isolated from kimchi were cultured by inoculating 3 μL into esculin medium, and strains showing brown rings were selected and used as the test material of the present invention.

Figure 112011005779419-pat00001

Figure 112011005779419-pat00001

김치에서 분리한 71종의 미생물을 상기 표 Esculin 배지에 3 μL를 접종하여 ß-glucosidase 생산균주를 선별하였다. Esculin은 미생물이 생성하는 ß-glucosidase에 의해 esculetin과 glucose로 분해되며, 분해된 esculetin은 철이온과 반응하여 진한 갈색 환을 형성하므로 실험결과 5종이 도 1과 같은 갈색 환을 나타내는 것을 확인하였다. 각각의 ß-glucosidase 활성을 측정한 결과, 분리 균주 KU244가 가장 높은 효소활성을 나타내었다. Ss-glucosidase producing strains were selected by inoculating 3 microliters of the 71 kinds of microorganisms isolated from Kimchi in the above-described Esculin medium. Esculin is decomposed into esculetin and glucose by the ß-glucosidase produced by the microorganism, and the decomposed esculetin reacted with iron ions to form a dark brown ring. As a result, five species showed brown rings as shown in FIG. As a result of measuring the ß-glucosidase activity, the isolated strain KU244 showed the highest enzyme activity.

본 발명의 상기 효모균주는 2010년 12월 29일자로 국제기탁기관 한국미생물보존센터(Korean Culture Center of Microorganisms, Seoul)에 기탁번호 KCCM 11158P로 기탁 되었다.
The yeast strain of the present invention was deposited on December 29, 2010 under the deposit No. KCCM 11158P at the International Depository Korean Culture Center of Microorganisms, Seoul.

균주의 동정Identification of the strain

ß-Glucosidase를 생산하는 KU 244 균주의 동정은 ㈜마크로젠(Seoul, Korea)에 18S rRNA 서열분석을 의뢰하여 동정하였다.Identification of KU 244 strain producing ß-Glucosidase was performed by requesting 18S rRNA sequencing from Macrogen (Seoul, Korea).

균주 동정을 위해서는 18S rRNA 서열분석 결과(도2), 99% 유의성으로 Saccharomyces servazzi로 동정되었으며, 분리 균주는 Saccharomyces servazzii KU 244로 명명하였다(도2).
For the identification of strains, 18S rRNA sequencing results (FIG. 2) were identified as Saccharomyces servazzi with 99% significance, and the isolated strain was named Saccharomyces servazzii KU 244 (FIG. 2).

균주의 배양 및 ß-Cultivation of strains and ß- glucosidaseglucosidase 활성측정 Activity measurement

본 발명 KU 244 균주는 lactobacilli MRS broth에 회분 배양한 후, 배양액 500 μl에 20% glycerol 500 μl를 혼합한 후 -70℃에서 동결하여 보관하였다. 배지는 lactobacilli MRS broth 10 mL, 본배양 배지는 lactobacilli MRS broth 100 ml를 121℃에서 15분 동안 autoclave하여 사용하였으며, 전배양은 24시간 동안 35℃에서 정치배양하고, 본배양은 전배양액 1%(v/v)를 접종하여 35℃, 72시간 동안 정치배양하였다. The KU 244 strain of the present invention was incubated in lactobacilli MRS broth, and then mixed with 500 μl of culture medium and 500 μl of 20% glycerol, and stored at -70 ° C. for freezing. The medium was lactobacilli MRS broth 10 mL, the main culture medium was 100 ml lactobacilli MRS broth autoclave at 121 ℃ for 15 minutes, the pre-culture was incubated at 35 ℃ for 24 hours, the main culture was 1% (preculture) v / v) was inoculated and incubated at 35 ° C. for 72 hours.

효소활성은 Kohchi 법(Kohchi et al., 1986)을 사용하여 측정하였다. 0.05 M sodium phosphate buffer(pH 5.5)에 녹인 5 mM pNPG(ρ-nitrophenyl-α-D-glucopyranoside) 1 mL와 배양상등액 1ml를 혼합한 후, 50℃에서 10분간 반응시켰다. 0.5 M Na2CO3 1 mL를 가하여 반응을 정지시킨 후, Spectrophotometer를 이용하여 400 nm에서 발색 정도를 측정하였다. 표준곡선은 4-nitrophenol을 사용하였으며, 효소활성은 1분 동안 상등액 1 mL가 pNPG를 분해하는 정도를 나타내었다.
Enzyme activity was measured using the Kohchi method (Kohchi et al., 1986). 1 mL of 5 mM pNPG (ρ-nitrophenyl-α-D-glucopyranoside) dissolved in 0.05 M sodium phosphate buffer (pH 5.5) and 1 ml of the culture supernatant were mixed and allowed to react at 50 ° C for 10 minutes. After the reaction was stopped by adding 1 mL of 0.5 M Na 2 CO 3 , the color development was measured at 400 nm using a spectrophotometer. The standard curve was 4-nitrophenol, and the enzyme activity showed that 1 mL of supernatant decomposed pNPG for 1 minute.

균주 배양시간에 따른 ß-Ss- according to strain incubation time glucosidaseglucosidase 활성 activation

배양시간, 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 48, 72 시간에 배양액 1 mL를 샘플링하고 생균수는 일반 도말법으로, 흡광도는 Spectrophotometer를 이용하여 630 nm에서 측정하였고, ß-glucosidase의 활성은 12,000 rpm, 10분 간 원심분리한 후 배양상등액을 취해 측정하였다.Sample 1 mL of the culture at incubation time, 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 48, 72 hours, viable cell count using normal smearing, and absorbance using Spectrophotometer. The ß-glucosidase activity was measured by centrifugation at 12,000 rpm for 10 minutes, followed by culture supernatant.

본 발명 신규한 효모 Saccharomyces servazzii KU244균주는 배양한지 6시간 후에 대수증식기를 나타냈으며, 배양 20시간 후에 정지기에 도달하는 것을 볼 수 있었다(도3). 정지기에서 생균수는 8.77 Log CFU/ml, 흡광도는 1.8 정도였으며 그 이후 50시간 까지 균수는 유지되었다. 효소활성은 미생물 성장곡선과 유사한 경향을 보였다. 균 접종 6 시간 이후로 효소활성이 꾸준히 증가하였으며, 24 시간에 17.73 μM/min·mL를 나타내었다. 균이 정지기가 지속되는 동안, 50 시간 배양까지도 효소활성 (19.41 μM/min·mL)은 소실되지 않고 조금씩 증가하는 것을 확인하였다.
The present invention novel yeast Saccharomyces servazzii Strain KU244 showed a logarithmic growth stage 6 hours after incubation, reaching the stop phase 20 hours after incubation (Fig. 3). In the stationary phase, the viable cell count was 8.77 Log CFU / ml and the absorbance was about 1.8. Enzyme activity showed similar trend with microbial growth curve. Enzyme activity increased steadily after 6 hours of inoculation and at 24 hours 17.73 μM / min · mL was shown. It was confirmed that the enzyme activity (19.41 μM / min · mL) increased little by little even after 50 hours of incubation, while the bacterium remained stationary.

반응온도에 따른 ß-Ss- according to reaction temperature glucosidaseglucosidase 활성 측정 Active measurement

0.05 M sodium phosphate buffer(pH 5.5)에 녹인 5 mM pNPG 1 ml와 배양상등액 1 ml를 혼합한 후, 반응 온도를 20, 30, 35, 40, 45, 50, 55, 60℃로 각각의 온도에서 10분 간 반응시켰다. 0.5 M Na2CO3 1 ml를 가하여 반응을 정지시킨 후, 효소활성을 측정하였다. After mixing 1 ml of 5 mM pNPG dissolved in 0.05 M sodium phosphate buffer (pH 5.5) and 1 ml of the culture supernatant, the reaction temperature was 20, 30, 35, 40, 45, 50, 55 and 60 ° C at each temperature. The reaction was carried out for 10 minutes. After the reaction was stopped by adding 1 ml of 0.5 M Na 2 CO 3 , the enzyme activity was measured.

반응 온도에 따른 ß-glucosidase의 효소 활성을 확인하기 위해, 반응 온도 20, 30, 35, 40, 45, 50, 55, 60℃로 각각 반응시켜 활성을 측정하였다. 20-50℃까지 온도가 올라갈수록 효소활성이 높게 나타났으며, 50℃에서 상등액 1 mL의 효소활성이 13.04 μM/min·mL을 나타내며 최대값을 나타냈다. 그러나 55℃ 이상에서 효소활성이 0.63 μM/min·mL로 급격히 감소하는 것을 볼 수 있었다(도4).
In order to check the enzyme activity of ß-glucosidase according to the reaction temperature, Activity was measured by reaction at the reaction temperature of 20, 30, 35, 40, 45, 50, 55, 60 degreeC, respectively. Enzyme activity was higher with increasing temperature up to 20-50 ℃, and the enzyme activity of 1 mL of supernatant at 50 ℃ was 13.04 μM / min · mL and showed the maximum value. However, the enzyme activity was rapidly reduced to 0.63 μM / min · mL above 55 ℃ (Fig. 4).

한국미생물보존센터(국외)Korea Microorganism Conservation Center (overseas) KCCM11158KCCM11158 2010122920101229

Claims (1)

김치유래의 ß-glucosidase 생산 효모균주 Saccharomyces servazzii KU 244(기탁번호 KCCM 11158P).Kimchi-derived ß-glucosidase-producing yeast strain Saccharomyces servazzii KU 244 (Accession No. KCCM 11158P).
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US20090035811A1 (en) 2007-07-31 2009-02-05 Kabushiki Kaisha Toyota Chuo Kenkyusho Artificial scaffolding material for protein retention and use of the same
JP2010154805A (en) 2008-12-26 2010-07-15 Shinshu Univ Method for producing bioethanol from water-soluble polysaccharide
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US20090035811A1 (en) 2007-07-31 2009-02-05 Kabushiki Kaisha Toyota Chuo Kenkyusho Artificial scaffolding material for protein retention and use of the same
JP2010154805A (en) 2008-12-26 2010-07-15 Shinshu Univ Method for producing bioethanol from water-soluble polysaccharide
KR20100131208A (en) * 2009-06-05 2010-12-15 고려대학교 산학협력단 A recombinant vector and a transformant with an improved productivity of bio-ethanol

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020004858A1 (en) * 2018-06-25 2020-01-02 (주)코엔바이오 Diabetes-alleviating or antioxidant composition comprising yeast extract and method for preparing yeast extract

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