구름송편버섯 자실체의 열수와 메탄올 추출물의 아미노산 성분

최병용 ( Byeong Yong Choi ),이형환 ( Hyung H. Lee ) 한국자연치유학회 2017 Journal of Naturopathy Vol.6 No.1-2

구름송편버섯 자실체를 열수(hot water)와 메탄올(methanol)에서 추출하여 추출물의 수율 및 유리 아미노산류와 유기산류의 함량을 조사 분석하였다. 구름송편버섯 자실체 열수 추출물 수율은 0.495 g, 메탄올 추출물은 0.246 g이었으며, 수율은 열수추출이 메탄올 추출보다 약 2배 높았다. 열수추출물에 함유된 유리 아미노산은 총 15종(총 13.59 mg/g)이었고, 이중 leu(2.1 mg/g)이 가장 높았고, arg(1.83 mg/g), ile(1.17 mg/g), tyr(0.03 mg/g) 순이었다. 필수아미노산 함량은 열수 추출물이 메탄올 추출물의 함량보다 약 41배 높았으나, lys은 존재하지 않았다. 메탄올 추출물의 유리 아미노산은 총 6종(총 0.33 mg/g)이었고, 이중 glu이 0.12 mg/g으로 가장 높게 나타났다. 메탄올 추출물에서는 필수아미노산 함량이 총 0.12 mg/g이었다. 유기산 물질의 열수추출은 7종(총 1.26 mg/g)이었고, 이중 γ-aminobutyric acid가 0.63 mg/g, phosphoserine이 0.21 mg/g 이었다. 메탄올 추출물에서의 유기물은 phosphoserine과 β-amino-isobutyric acid의 2종이 확인되었으며, 총 0.09 mg/g이었다. 이중 β-amino-isobutyric acid는 열수 추출물에는 존재하지 않았으나, 메탄올 추출물에서는 확인이 되었다. 구름송편버섯 자실체 열수와 메탄올 추출물에는 여러 종류의 아미노산과 유기산이 추출되어서 단백질의 아미노산 기능과 유기산 등의 기능이 다양한 영양학적 또는 기능성 원료로서의 활용 가능성이 있는 것으로 사료된다. This study was to analyze, using HPLC method, the quantity and types of L-amino acids and organic compounds found in Coriolus versicolor fruitbody extracts in hot water and methanol. The total quantity of the C. versicolor extracts was 0.495 g in the hot water, the methanol extract appeared to 0.246 g, the water extracts showed about two times higher than the content of methanol extract. Fifteen types of L-amino acids and seven kinds of organic acids were found in the both extracts. The total quantity of the free amino acids were 13.59 mg/g in the hot water extracts, and the amounts of leu was highest with 2.1 mg/g, and the rest ranged from arg(1.83 mg/g), ile(1.17 mg/g) and tyr(0.03 mg/g). The total quantity of the free amino acids were 0.33 mg/g and six types of amino acids in methanol extracts, and the amounts of glu was highest with 0.12 mg/g, and the rest ranged from arg(1.83 mg/g), ile(1.17 mg/g) and tyr(0.03 mg/g). Amounts of essential amino acids were 41 times more presented in the hot extracts than the methanol extracts, however lys was not in the hot extract. The hot extracts contained seven types of organic acids and total quantity was 1.26 mg/g; ?-aminobutyric acid was 0.63 mg/g, phosphoserine was 0.21 mg/g. The methanol extract contained phosphoserine and β-amino-isobutyric acid and total amounts was 0.09 mg/g. β-amino-isobutyric acid was in the hot water. The finding supports that C. versicolor fruitbody extracts from hot water and methanol are useful to basic information in nutritional and functional materials.

Production of chiral β-amino acids using ω-transaminase from Burkholderia graminis

Mathew, S.,Bea, H.,Nadarajan, S.P.,Chung, T.,Yun, H. Elsevier Science Publishers 2015 Journal of biotechnology Vol.196 No.-

<P>Optically pure beta-amino acids are of high pharmacological significance since they are used as key ingredients in many physiologically active compounds. Despite a number of enzymatic routes to these compounds, an efficient synthesis of beta-amino acids continues to pose a major challenge for researchers. omega-Transaminase has emerged as an important class of enzymes for generating amine compounds. However, only a few omega-transaminases have been reported so far which show activity towards aromatic beta-amino acids. In this study, (S)-omega-transaminase from Burkholderia graminis C4D1M has been functionally characterized and used for the production of chiral aromatic beta-amino acids via kinetic resolution. The enzyme showed a specific activity of 3.1 U/mg towards rac-beta-phenylalanine at 37 degrees C. The K-m and K-cat values of this enzyme towards rac-beta-phenylalanine with pyruvate as the amino acceptor were 2.88 mM and 91.57 min(-1) respectively. Using this enzyme, racemic beta-amino acids were kinetically resolved to produce (R)-beta-amino acids with an excellent enantiomeric excess (> 99%) and similar to 50% conversion. Additionally, kinetic resolution of aromatic beta-amino acids was performed using benzaldehyde as a cheapamino acceptor. (C)2015 Elsevier B.V. All rights reserved.</P>

아미노산 첨가가 꽃송이버섯 균사체 성장 및 베타글루칸, GABA 함량 변화에 미치는 영향

조한교,신현재 한국버섯학회 2017 한국버섯학회지 Vol.15 No.1

Sparassis latifolia (formerly S. crispa) is used in food and nutraceuticals or dietary supplements, as rich in flavor compounds and β-glucan. Some previous studies have reported the effects of mushroom on brain function, including its neuroprotective effect. Thus, for this mushroom to be used as an effective nutraceutical for brain function, it would be desirable for it to contain other compounds such as γ-aminobutyric acid (GABA) in addition to β-glucan. In this study, the enhancement of growth and GABA production in the mycelium of medicinal and edible mushroom S. latifolia was investigated. Amino acids were added externally as the main source of nutrition, and the effects of amino acids were investigated using liquid medium, specifically amino acid-free potato dextrose broth (PDB). The amino acids added were L-glutamic acid (named PDBG medium) and L-ornithine (named PDBO medium). The growth of mycelia was determined to be 0.9 ± 0.00 g/L, 2.2 ± 0.16 g/L, and 1.93 ± 0.34 g/L PDBG respectively. The GABA content was 21.3 ± 0.9 mg/100 g in PDB medium, and it in PDBG 1.4% medium, at 115.4 ± 30.2 mg/100 g. However, the PDBO medium was not effective in increasing the GABA content of mycelia. Amino acids had little effect on the β-glucan content of mycelia. The β-glucan content was 39.7 ± 1.4 mg/100 mg, 34.4 ± 0.2 mg/100 mg, and 35.2 ± 9.2 mg/100 mg in PDB, PDBG 1.8% and PDBO 1.4% media, respectively. Addition of glutamic acid and ornithine positively affected the growth of S. latifolia mycelia, and glutamic acid positively affected GABA production; no degradation of GABA was observed with addition of glutamic acid.

화학적 수식에 의한 Bacillus stearothermophilus β-D-Xylosidase의 연구

서정한,최용진 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.6

E. coli JM109/pMG1 재조합 균주가 생산하는 Bacillus stearothermophilus β-Dxylosidase의 효소활성과 관련된 필수 아미노산 잔기를 화학적 수식(chemical modification) 법을 이용, 분석하여 다음과 같은 결과를 얻었다. B. stearothermophilus β-D-xylosidase의 구성 아미노산 잔기 중에서 histidine, tyrosine, serine, methionine 및 tryptophan 등은 효소 활성에 직접적인 관계를 가지고 있지 않는 것으로 판명되었으며 carboxylic amino acid와 cysteine은 효소 작용에 직접 관련된 중요한 역할을 하고 있는 아미노산 잔기로 밝혀졌다. 한편 xylobiose 기질에 의한 효소 활성 보호 실험결과에 의하면 상기 두 종의 효소 활성 필수 아미노산 잔기는 효소-기질 결합 단계에서 중요한 역할을 하는 것으로 추측되었다. 또한 각각 carboxylic amino acid와 cysteine modifier인 Woodward's Reagent K(WRK)와 N-ethylmaleimide의 효소 불활성화 반응의 kinetics를 분석해 본 결과 WRK의 경우에는 두개의 carboxylic amino acid를, N-ethylmaleimide의 경우는 한개의 cysteine 잔기를 수식함으로써 거의 완전한 효소 불활성화 현상을 초래하는 것으로 계산되었다. Essential amino acids involving in the catalytic mechanism of the β-D-xylosidase of Bacillus stearothermophilus were determined by chemical modification studies. Among various chemical modifiers tested N-bromosuccinimide (NBS), p-hydroxymercurybenzoate (PHMB), N-ethylmaleimide, 1-[3-di-ethylamino)-propy1]-3-ethylcarbodi-imide(EDC), and Woodward's Reagent K(WRK) inactivated the enzyme, resulting in the residual activity of less than 20%. WRK reduced the enzyme activity by modifying carboxylic amino acids, and the inactivation reacion proceeded in the form of pseudo-first-order kinetics. The double-lagarithmic plot of the observed pseudo-first-order rate constant against the modifier concentration yielded a reaction order of 2, indicating that two carboxylic amino acids were essential for the enzyme activity. The β-D-xylosidase was also inactivated by N-ethylmaleimide which specifically modified a cysteine residue with a reaction order of 1, implying that one cysteine residue was important for the enzyme activity. Xylobiose protected the enzyme against inactivation by WRK and N-ethylmaleimide, revealing that carboxylic amino acids and a cysteine residue were present at the substrate-binding site of the enzyme molecule.

Resolution of β-amino acids on a high performance liquid chromatographic doubly tethered chiral stationary phase containing N–CH₃ amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

Hyun, Myung Ho,Song, Yanci,Cho, Yoon Jae,Choi, Hee Jung WILEY-VCH Verlag 2007 Journal of separation science Vol.30 No.15

<P>A doubly tethered chiral stationary phase (CSP) containing N–CH<SUB>3 </SUB>amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of various β-amino acids. The chiral recognition behaviors for the resolution of β-amino acids on the doubly tethered CSP were consistent with those on the corresponding singly tethered CSP while the chiral recognition ability of the doubly tethered CSP was generally greater in terms of both the separation (α) and the resolution factors (R<SUB>S</SUB>) than that of the corresponding singly tethered CSP. From these results, it was concluded that attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the CSP improves the chiral recognition ability for the resolution of β-amino acids without any change in the chiral recognition mode. The retention factors (k<SUB>1</SUB>) on the doubly tethered CSP were larger than those on the corresponding singly tethered CSP and these retention factors were found to be controllable with the variation of the type and the content of the organic and/or acidic modifier in the aqueous mobile phase without significant change in the separation and the resolution factors.</P>

주요 식용버섯의 추출용매에 따른 생리활성 및 아미노산 성분 차이 비교

안기홍,한재구,조재한 한국버섯학회 2020 한국버섯학회지 Vol.18 No.1

본 연구에서는 큰느타리, 느타리, 팽이, 표고, 양송이를 대상으로 건강기능성 식품과 김치, 장아찌 등의 저장식품 개발의 기초자료로 활용하고자 30%와 70% 발효주정 및 5%, 10%, 15% NaCl 조건에서 추출물 내의 생리활성 성분과 아미노산 성분함량의 차이를 알아보기 위하여 실험을 수행하였다. 각 버섯의 추출용매별 생리활성 성분분석 결과, DPPH 라디컬 소거능은 큰느타리와 느타리의 30% 발효주정 추출물에서 가장 높은 소거능을 보였다. 아질산 염소거능은 큰느타리와 느타리의 70% 발효주정 추출물에서 소거능이 높았으며, 팽이 및 표고, 양송이는 30% 발효주정 추출물이 가장 높았다. 총 폴리페놀 함량의 경우 큰느타리는 30% 발효주정 추출물에서, 느타리는 70% 발 효주정 추출물에서 높은 함량치를 보였으며 팽이는 70% 발효주정 추출물에서, 표고와 양송이의 경우 30% 발효주정 추출물에서 가장 높은 총 폴리페놀 함량을 보였다. 각 추출용매별 아미노산 분석결과, 총 아미노산 함량은 큰느타리가 가장 높았으며 70% 발효주정 추출물에서 가장 많은 양이 검출되었다. 버섯 별로 큰느타리의 경우 70% 발효주정 추출물에서 프롤린(Pro)과 페닐알라닌(Phe)이 가장 많이 검출되었으며, 느타리는 글루탐산(Gle) 함량이 다른 아미노산 성분들에 비하여 월등히 높았다. 팽이는 글루탐산(Glu), 알라닌(Ala), 프롤린(Pro)이 가장 높은 함량을 보였으며, 표고는 글루탐산(Glu)이 가장 높은 함량치를 보였고, 양송이는 5% NaCl 염추출물에서 글루탐산 (Glu)과 알라닌(Ala) 함량이 높게 검출되었다. 위와 같은 결과로부터 항산화와 관련된 생리활성 성분은 발효주정 추출물에서 높았으며, 아미노산 함량은 버섯의 종류, 품종 또는 추출용매 별로 아미노산 성분함량의 차이가 나타나 향후 용도에 따라 더욱 세부적으로 검토할 필요가 있다. The aims of this study were to investigate the biological activities and amino acid contents of fermentation ethanol and sodium extracts from five edible mushrooms (Pleurotus eryngii, Pleurotus ostreatus, Flammulina velutipes, Lentinula edodes, and Agaricus bisporus). DPPH radical scavenging activities in 30% fermentation ethanol extracts of P. eryngii and P. ostreatus were significantly higher than those in sodium extracts (p<0.05). Nitrite scavenging activities were also higher in the 30% fermentation ethanol extracts of P. eryngii and P. ostreatus. The total polyphenol contents of P. eryngii, P. ostreatus, and F. velutipes were high in 70% fermentation ethanol extracts. The analysis of amino acids revealed that the 70% fermentation ethanol extract of P. eryngii had the highest content of total amino acids, with higher phenylalanine, leucine, isoleucine, valine, and tyrosine contents higher than the other extracts. In all the extracts of P. ostreatus, glutamic acid was the most abundant amino acid. The 5% NaCl and 30% fermentation ethanol extracts of F. velutipes contained abundant glutamic acid, alanine, and proline. Glutamic acid was the most abundant amino acid in the 70% and 30% fermentation ethanol extracts of L. edodes. In the 5% NaCl extracts of A. bisporus, glutamic acid and alanine were abundant. Thus, maximum biological and nutritional ingredients can be extracted using the optimal solvents for each type of mushroom.

Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase

Kim, Geon-Hee,Jeon, Hyunwoo,Khobragade, Taresh P.,Patil, Mahesh D.,Sung, Sihyong,Yoon, Sanghan,Won, Yumi,Choi, In Suk,Yun, Hyungdon Elsevier 2019 Enzyme and microbial technology Vol.120 No.-

<P><B>Abstract</B></P> <P>Enantiopure β-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model β-amino acid (<I>R</I>)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted β-ketoester substrate to β-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding β-amino acid. A potent enzyme from <I>Ilumatobacter coccineus</I> (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM β-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% <I>ee</I>). Furthermore, ω-TAIC was successfully used for the synthesis of various β-amino acids from their corresponding β-keto ester substrates.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A potent ω-TA was identified using bioinformatic tools from 16 potential enzymes. </LI> <LI> 3-ATfBA was produced with good conversion (81.9%) and 99% <I>ee.</I> </LI> <LI> ω -TAIC was successfully used for the synthesis of various β-amino acids. </LI> <LI> Lowering the reaction pH to 7.0 can circumvent the inhibition of ω -TAIC by 3-ATfBA. </LI> </UL> </P>

Amino acid digestibility in diets containing copra meal with β-mannanase fed to growing pigs

Jang Jae Cheol,Kim Dong Hyuk,Jang Young Dal,김유용 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.12

Objective: The objective of this study was to estimate standardized ileal digestibility (SID) of amino acids (AA) in growing pigs fed diets containing increasing levels of copra meal (CM) with β-mannanase supplementation.Methods: Twenty barrows (initial body weight: 34.43±0.11 kg) surgically fitted with T-cannulas at the distal ileum were individually housed in metabolism crates. Pigs were allotted to 5 dietary treatments in a completely randomized design with 4 replicates per treatment. The dietary treatments were: i) NC, negative control, corn-soybean meal (SBM) based diet, ii) PC, positive control, basal diet + 0.10% β-mannanase supplementation (800 IU/kg), iii) CM6, PC diet with 6% CM supplementation, iv) CM12, PC diet with 12% CM supplementation, and v) CM18, PC diet with 18% CM supplementation. A nitrogen-free diet was used to estimate basal endogenous losses of AA for SID calculation. All experimental diets contained 0.5% chromic oxide as an indigestible marker. Each period consisted of a 4-d diet adaptation period and a 3-d ileal digesta collection period.Results: There were no differences in apparent ileal digestibility (AID) and SID of all AA between the NC and PC treatments except that the PC treatment had lower AID and SID of glycine than the NC treatment (p<0.05). There were linear decreases in AID and SID of lysine (p<0.05) and aspartic acid (p = 0.06; tendency) with increasing levels of CM in the diets with β-mannanase.Conclusion: The β-mannanase supplementation had no effect on AA digestibility in pigs fed the corn-SBM based diet but increasing levels of CM reduced SID of lysine and aspartic acid. Objective: The objective of this study was to estimate standardized ileal digestibility (SID) of amino acids (AA) in growing pigs fed diets containing increasing levels of copra meal (CM) with β-mannanase supplementation. Methods: Twenty barrows (initial body weight: 34.43±0.11 kg) surgically fitted with T-cannulas at the distal ileum were individually housed in metabolism crates. Pigs were allotted to 5 dietary treatments in a completely randomized design with 4 replicates per treatment. The dietary treatments were: i) NC, negative control, corn-soybean meal (SBM) based diet, ii) PC, positive control, basal diet + 0.10% β-mannanase supplementation (800 IU/kg), iii) CM6, PC diet with 6% CM supplementation, iv) CM12, PC diet with 12% CM supplementation, and v) CM18, PC diet with 18% CM supplementation. A nitrogen-free diet was used to estimate basal endogenous losses of AA for SID calculation. All experimental diets contained 0.5% chromic oxide as an indigestible marker. Each period consisted of a 4-d diet adaptation period and a 3-d ileal digesta collection period. Results: There were no differences in apparent ileal digestibility (AID) and SID of all AA between the NC and PC treatments except that the PC treatment had lower AID and SID of glycine than the NC treatment (p<0.05). There were linear decreases in AID and SID of lysine (p<0.05) and aspartic acid (p = 0.06; tendency) with increasing levels of CM in the diets with β-mannanase. Conclusion: The β-mannanase supplementation had no effect on AA digestibility in pigs fed the corn-SBM based diet but increasing levels of CM reduced SID of lysine and aspartic acid.

Thermostable Ω-Transaminase From Sphaerobacter Thermophilus and Its Application For Producing Aromatic Β- and Γ-Amino Acids.

Hyunwoo JEon,Sam MATHEW,SP NADARAJAN,Hyungdon YUN 한국생물공학회 2017 한국생물공학회 학술대회 Vol.2017 No.4

Effect of Ammonia on Anaerobic Degradation of Amino Acids

박세용,김문일 대한토목학회 2016 KSCE JOURNAL OF CIVIL ENGINEERING Vol.20 No.1

Since amino acids are the main intermediates converted from protein and they produce ammonia which affects on the efficiency of anaerobic digestion, the effect of ammonia on anaerobic degradation of the amino acids should be evaluated. As the initial NH4 +-N concentrations increased from 0 to 5,000 mg NH4 +-N/L, deamination of the amino acids decreased from 100 to 55% implying that deamination was inhibited by the ammonia toxicity. Although the initial NH4 +-N was absent, the produced ammonia from the amino acids inhibited methane production. VFAs produced through deamination accumulated and were not converted to methane due to the ammonia toxicity. Among all of the amino acids, only β-alanine was very sensitive to ammonia inhibition. Conclusively, concentration of ammonia both in wastewater and to be produced from amino acids should be investigated for better anaerobic digestion of wastewater having a high content of protein such as hydrolysed wastewater sludge.