Enhancing the Enzymatic Activity of the Multifunctional β-Glycosyl Hydrolase (Cel44C-Man26A_P558) from Paenibacillus polymyxa GS01 Using DNA Shuffling

강영민,강태호,윤한대,조계만,Kang, Young-Min,Kang, Tae-Ho,Yun, Han-Dae,Cho, Kye-Man The Microbiological Society of Korea 2012 미생물학회지 Vol.48 No.2

본 연구자들은 이전에 cellulase, xyalnase 및 lichenase의 다기능 효소활성을 지니는 절단된 Cel44C-$Man26A_{P558}$의 ${\beta}$-glycosyl hydrolase를 보고하였다. 본 연구에서는 절단된 Cel44C-$Man26A_{P558}$ 효소의 다기능성 ${\beta}$-glycosyl hydrolase 활성을 증가시키기 위해 DNA shuffling을 시도하였다. DNA shuffling에 의해 단일변이(P438A)를 가진 M2Cel44C-$Man26A_{P558}$와 이중변이(A273T 및 P438A)를 가진 M21Cel44C-$Man26A_{P558}$를 얻었다. 이중변이를 가진 M21Cel44C-$Man26A_{P558}$은 단일변이를 가진 M2Cel44C-$Man26A_{P558}$ 보다 효소활성이 낮게 나타났으나, M2Cel44C-$Man26A_{P558}$와 M21Cel44C-$Man26A_{P558}$은 대조구인 Cel44C-$Man26A_{P558}$ 보다 약 1.3에서 2.2배 정도 높은 효소활성을 나타내었다. 특히, 단일변이를 가진 M2Cel44C-$Man26A_{P558}$는 대조구인 Cel44C-$Man26A_{P558}$보다 cellulase, xylanase 및 lichenase 효소활성이 약 1.5에서 2.2배 정도 높게 나타났다. ${\beta}$-Glycosyl hydrolase의 cellulase, linchenase 및 xylanase 최적 효소활성은 각각 pH 7.0, 7.0 및 6.0에서 이었다. 이러한 결과는, 아미노산 잔기인 Ala438이 다기능성 ${\beta}$-glycosyl hydrolase 활성을 증가시키는 중요한 역할을 한다고 추정할 수 있다. We previously reported that the truncated Cel44C-$Man26A_{P558}$ ${\beta}$-glycosyl hydrolase protein exhibits multifunctional activities, including cellulase, xylanase, and lichenase. DNA shuffling of the truncated Cel44C-$Man26A_{P558}$ enzyme was performed to enhance the enzymatic activity of the multifunctional ${\beta}$-glycosyl hydrolase. Two mutant enzymes, M2Cel44C-$Man26A_{P558}$ that carries one mutation (P438A) and M21Cel44C-$Man26A_{P558}$ that carries two mutations (A273T and P438A) were obtained. The enzymatic activity of the M21Cel44C-$Man26A_{P558}$ double mutant was lower than enzymatic activity of the single mutant (M2Cel44C-$Man26A_{P558}$). However, both mutants displayed the enhancements in their enzyme activities that were ${\approx}1.3$- to 2.2-fold higher than the original enzymatic activity in Cel44C-$Man26A_{P558}$. In particular, the mutant M2Cel44C-$Man26A_{P558}$ exhibited an approximate 1.5- to 2.2-fold increase in the cellulase, xylanase, and lichenase activities in comparison with the control (Cel44C-$Man26A_{P558}$). The optimum cellulase, linchenase, and xylanase activities of ${\beta}$-glycosyl hydrolase were observed at pH 7.0, pH 7.0 and pH 6.0, respectively. These results, therefore, suggest that the amino acid residue Ala438 plays important roles in the enhancement of the activity of multifunctional ${\beta}$-glycosyl hydrolase.

볶음처리에 의한 자색고구마의 항산화 증진 효과

조계만 ( Kye Man Cho ),주옥수 ( Ok Soo Joo ) 한국식품저장유통학회(구 한국농산물저장유통학회) 2012 한국식품저장유통학회지 Vol.19 No.5

Purple sweet potato (PSP, Ipomoea batatas) has various biological effects including antidiabetic, anti-inflammatory, and antioxidant activities. In this study, the antioxidant activity of PSP after roasting were compared using DPPH, ABTS, and FRAP assays. In addition, the total phenolics and flavonoid contents, Maillard reaction products, and phenolic acid contents were measured to identify the factors that changed PSP,s antioxidant activity due to roasting. The roasted PSP exhibited significantly higher antioxidant activity than unroasted PSP. In particular, the PSP roasted at 200℃ for 10 min showed the highest antioxidant activity among all the PSPs that were roasted under different conditions. The total phenolic and flavonoid contents, Maillard reaction products and phenolic acid contents markedly increased, corresponding to the general increase in antioxidant activities after roasting. These results suggest that roasted PSP extracts are potential source of natural antioxidants that may be used in certain food applications.

약초 추출액을 사용하여 제조한 감주의 젖산발효

조계만(Kye Man Cho),안병용(Byung Yong Ahn),서원택(Weon Taek Seo) 한국식품과학회 2008 한국식품과학회지 Vol.40 No.6

한방발효음료를 제조하기 위하여 한방감주의 젖산발효를 유도하였다. 이를 위해 각종 시료로부터 다양한 젖산균을 순수 분리하여 발효적성을 검토한 결과 최적균주로서 LAB19 균주를 선별하였다. LAB19 균주는 곶감으로부터 분리하였으며 생리 화학적 특성 및 16S rRNA 염기서열 분석을 통하여 Leuconostoc mesenteroides로 동정되었다. 한방감주에 종배양한 LAB19 균주를 2.5%(v/v) 접종하고 25oC에서 60시간 발효시켰을 때, 한방감주는 141.3 g/L의 환원당과 5.33 g/L의 유기산, 그리고 1.19 g/L의 가용성 페놀을 함유하고 있었다. 당과 유기산 구성을 살펴보면 당의 약 90%는 맥아당이었으며, 유기산의 58%는 젖산이었다. 수용성 phenolics 성분 등에 기인하는 라디칼 소거 활성은 L-ascorbic acid의 92.4% 보다 낮은 76.6-75.7% 범위를 유지하고 있었다. In this study, the characteristics of the lactic fermentation of gamju manufactured using a medicinal herb decoction were assessed. A bacterial strain, LAB19, which is used for the induction of lactic fermentation into gamju, was isolated from dried persimmon and identified as Leuconostoc mesenteroides on the basis of morphological, physiological, and chemotaxonomical features, and 16S rRNA sequencing analysis. After 60 hours of lactic fermentation with Leuconostoc mesenteroides LAB19 at 25℃, the gamju was determined to contain 141.3 g/L of reducing sugar, 5.33 g/L of acids, and 1.19 g/L of soluble phenolics. Approximately 90% of reducing sugar and 58% of acids were maltose and lactic acid, respectively. Free radical scavenging activities were retained at levels between 76.6 to 75.7% during the lactic fermentation of gamju.

단감(Diospyros kaki, T) 와인 제조에 관한 연구

조계만(Kye Man Cho),이정복(Jung Bock Lee),강군중(Goon Gjung Kahng),서원택(Weon Taek Seo) 한국식품과학회 2006 한국식품과학회지 Vol.38 No.6

옥수수 수염 추출액을 이용한 식혜 제조

조계만 ( Kye Man Cho ),주옥수 ( Ok Soo Joo ) 한국식품저장유통학회 ( 구 한국농산물저장유통학회 ) 2010 한국식품저장유통학회지 Vol.17 No.5

Acetobacter pasteurianus A8를 이용한 우리밀(금강밀) 식초 제조

조계만(Kye Man Cho),신지현(Ji Hyeon Shin),서원택(Weon Taek Seo) 한국식품과학회 2013 한국식품과학회지 Vol.45 No.2

우리밀(금강밀)을 기질로 사용한 식초 생산을 위하여 초산 생성능이 우수한 초산균을 재래 식초로부터 분리하여 A. pasteurianus A8으로 동정하였다. 우리밀 엿기름의 제조를 위해 발아 조건을 살펴본 결과, 15℃에서 6일간 발아 시킨 밀의 amylase 활성이 608.4 unit으로 가장 높았다. A. pasteurianus A8 균주를 종균으로 사용하여 우리밀 알코올 발효액의 초산 발효 조건을 살펴본 결과, 발효온도 30℃, 초기 알코올 농도 5.0%, 및 종균 접종량 5.0%에서 24일간 정치 발효하여 5.8%의 초산을 생산할 수 있었다. We tested the possibility of utilizing Korea domestic wheat (winter wheat variety “keumkangmil”) as a source of vinegar production. After saccharification of the whole-wheat flour with wheat malt, the saccharized liquid undergoes alcoholic fermentation, followed by acetic fermentation. Acetic acid bacterium A8, which showed the highest acetic acid production (4.56%) with domestic wheat as substrate, was selected from conventional vinegars. The strain A8 was identified as Acetobacter pasteurianus A8 through phylogenetic study using 16S rDNA sequencing analysis. The optimal condition for the malt enzyme was found to be 15℃ for germination periods of 6 days; its amylase activity was 608.4 U. Acetic acid production from domestic wheat substrate supplemented with 5% ethyl alcohol reached 5.8% after 24 days of static fermentation at 30℃ with a seeding rate of 5%.

Cloning of Isoamylase Gene of Pectobacterium carotovorum subsp. carotovorum LY34 and Identification of Essential Residues of Enzyme

조계만,김은주,레누카라디아마스,샤모허마드아스라풀,홍선주,김종옥,신기재,이영한,김훈,윤한대,Cho, Kye-Man,Kim, Eun-Ju,Math, Renukaradhya K.,Asraful Islam, Shah Md.,Hong, Sun-Joo,Kim, Jong-Ok,Shin, Ki-Jae,Lee, Young-Han,Kim, Hoon,Yun, Han-Dae Korean Society of Life Science 2007 생명과학회지 Vol.17 No.9

연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다. The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.

Changes in Phenolic Compounds (Isoflavones and Phenolic acids) and Antioxidant Properties in High-Protein Soybean (Glycine max L., cv. Saedanbaek)for Different Roasting Conditions

이진환,Kye Man Cho,이병원,Balo Kim,김현태,고종민,백인열,서원택,강영민,조계만 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.5

Contents of phenolic compound including isoflavones and phenolic acids as well as antioxidant effects in high-protein soybean cultivar “Saedanbaek” were evaluated under different roasting conditions. The roasted soybean exhibited significantly higher antioxidant activity than unroasted soybean in the three antioxidant methods including 2,2-diphenyl-1-picrylhydrazyl, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and Ferric reducing antioxidant power. In particular, the roasted soybean at 200oC for 15 min showed the highest antioxidant activity in comparison with other conditions. The contents of phenolic compounds, isoflavone aglycones (genistein, daidzein, and glycitein),isoflavone β-glucosides (genistin, daidzin, and glycitin), and phenolic acids increased, whereas isoflavone malonyl-β-glucosides decreased during roasting process. Moreover, total phenolic and flavonoid contents as well as those of isoflavone aglycones,isoflavone-β-glucosides, and phenolic acids increased, leading to a general increase in antioxidant activity after roasting. These results suggest that the roasting soybean extracts could contribute to obtaining natural antioxidants in certain food applications.

Identification of Auxin from Pseudomonas sp. P7014 for the Rapid Growth of Pleurotus eryngii Mycelium

강영민,조계만,Kang, Young Min,Cho, Kye Man The Microbiological Society of Korea 2014 미생물학회지 Vol.50 No.1

Pseudomonas sp. P7014 박테리아를 통한 큰느타리버섯 균사체의 생육촉진에 관한 연구가 수행되었다. 박테리아 배양액으로 부터 분리한 ethyl acetate 분획물(F5)에는 성장촉진물질(GPC)이 함유되어 있음을 확인하였다. TLC, HPLC, NMR 및 MS/MS분석법으로 확인한 바, indole acetic acid (IAA)로 확인되었다. 큰느타리버섯 균사체는 성장촉진물질(GPC)이 첨가된 PDA와 PDB 배지에서 빠른 성장을 보였다. 성장촉진물질(GPC)의 농도는 1.0 nM로 매우 낮았지만, 확인된 tryptophan은 IAA의 전구체로써 IAA가 아민화된 형태였다. 이들 결과는 박테리아에서 분비된 성장촉진물질(GPC)은 IAA이었고 큰느타리버섯 균사체의 생육촉진에 중요한 역할을 하는 것으로 확인되었다. The promoting effect of Pseudomonas sp. P7014 on the mycelia growth of Pleurotus eryngii was investigated. An ethyl acetate fraction (F5) from the culture supernatant of the bacteria was confirmed to contain the growth promoting compound (GPC). The GPC was identified to be indole acetic acid (IAA) by TLC, HPLC, MS/MS, and NMR analyses. P. eryngii mycelia grew rapidly both on PDA and in PDB after the treatment of GPC. The promoting concentration of GPC was as low as 1.0 nM. Tryptophan, the aminated form of IAA, was confirmed to be the precursor of IAA. These results suggested that bacterial secreted compound was IAA and plays an important role in promoting growth of mushroom mycelia.

Cloning and Identification of Essential Residues for Thermostable β-glucosidase (BgIB) from Thermotoga maritima

홍수영,조계만,김용희,홍선주,조수정,조용운,김훈,윤한대,Hong, Su-Young,Cho, Kye-Man,Kim, Yong-Hee,Hong, Sun-Joo,Cho, Soo-Jeong,Cho, Yong-Un,Kim, Hoon,Yun, Han-Dae Korean Society of Life Science 2006 생명과학회지 Vol.16 No.7

A hyperthermophilic bacterium Thernotoga maritima produced thermostable ${\beta}-glucosidase$. The gene encoding ${\beta}-glucosidase$ from T. maritima MSB8 was cloned and expressed in Escherichia coli. The en-zyme (BgIB) hydrolyzed ${\beta}-glucosidase$ linkages between glucose and alkyl, aryl of saccharide groups such as salicin, arbutin, and $_pNPG$. The insert DNA contained ORF with 2,166 bp encodes a 721 amino acids (calculated molecular mass of 80,964 and pl of 4.93). The amino a.id sequence of BglB showed the similarity to family 3 glycosyl hydrolases. The molecular weight of the enzyme was estimated to be approximately 81kDa by MUG-nondenaturing PAGE (4-methylumbelliferyl 13-D-glucoside-nondenaturing polyacrylamide gel electophoresis) and SDS-PACE. The ${\beta}-glucosidase$ exhibited maximal activity at pH 7.0 and $80^{\circ}C$. By exchanging two possible residues (Glu-232 and Asp-242) to Ala by site-directed mutagenesis method, it was found that these were essential for enzymatic activity. 초고온성 세균인 Thermotoga maritima로부터 ${\beta}-glucosidase$ 유전자를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 salicin, arbutin, $_pNPG$과 같은 탄소원의 ${\beta}$-글루코시드 결합을 가수분해하였다. 721개의 아미노산을 암호화하는 2,166 bp의 DNA 염기서열로된 유전자이였다. 다른 ${\beta}-glucosidase$ 효소들과 단백질 유사성을 비교한 결과 glycosyl hydrolase family 3에 속하였으며 MUG-nondenaturing PAGE와 SDS-PAGE에 의해 확인된 단백질의 크기는 약 81 kDa이었다. 효소활성은 pH 7.0, $80^{\circ}C$에서 가장 높은 활성을 나타냈으며 이 효소의 아미노산 서열에 있는 두 개의 아미노산 잔기 (232번 글루탐산과 242번 아스파르트산 잔기)를 알라닌으로 치환시켜 활성이 없어지는 것으로 보아 이 두 잔기가 효소활성에 중요한 역할을 하는 것으로 추정된다.