방사선 BY-445가 생성하는 α-amylase 저해물질의 특성

방병호,이진영 서울보건대학 1997 서울보건대학 부설 한국보건과학연구소 논문집 Vol.4 No.1

α-amylase에 대한 강력한 저해물질을 생산하는 방사균 BY-445를 토양으로부터 분리하였으며 이 균이 생산하는 배양액을 Amberlite IRA 420 column, Silica gel column chromatography 및 Sephadex G-50 column을 이용하여 조정제하였다. 이때 수율은 21.4%였으며 비활성도는 6배 증가하였다. 그리고 저해제의 α-amylase 100㎍에 대해 2배의 농도인 200㎍에서 저해활성이 거의 95%로 최고에 달하였다. 본 저해물질의 활성에 미치는 금속이온의 영향으로 Co^(+2), Hg^(+2), Pb^(+2), 및 Cu^(+2) 등에 의해 상대활성이 각각 13, 30, 37 및 72%로 각각 나타났다. 또한 본 저해물질은 열에 대한 안정성이 커서 100℃에서 120분간의 열처리를 하여도 그 활성은 거의 잔존하였으며 또한 전 pH 범위(pH2.0~12.0)에서도 아주 안정하였다. 본 저해물질의 여러가지 생화학적 반응을 검토한 결과 Anthrone, Molisch, Phenol-sulfuric acid반응에서는 양성을 띠었으며, Ninhydrin, Biuret, Benedict 등의 반응에서는 음성으로 나타났으며, 각종 carbohydrases에 대한 본 저해물질의 활성을 검토한 결과 사람 침 α-amylase와 Bacillus subtilis,porcine pancreas,Asp.niger의 glucoamylase등의 α-amylase에 대한 저해율이 각각 87%와 81,79,63%로 나타났으며, Aspergillus속의 cellulase와 Saccharomyces cerevisiae의 invertase에 대한 저해는 나타나지 않았다. A strain of Actinomycetes BY-445 isolated from soil was able to produce a biological active substance that has a strong inhibitory activity against hydrolysis by α-amylase. Extracellular inhibitory substance (inhibitor) from the culture broth of Actinomycetes BY-445 was purified to partial homogeneity by procedures including Amberlite IRA 420 column, Silica gel column, and Sephadex G-50 column chromatography. The partial purifying procedures resulted in 6-fold purification with the overall yield of 21.4% and the velocity of the inhibitor was reached at maximum in inhibitor concentration of 200㎍ against α-amylase enzyme concentration of 100㎍. In effect of metal salt, Co^(+2), Hg^(+2), Pb^(+2) and Cu^(+2), the relative inhibitory activity were shown about 13, 30, 37 and 72%, respectively and the inhibitor was also appeared to be relatively thermostable, and no appreciable inactivation was observed after incubation at 100℃ for 2 hour. The substance was stable in pH range from 2.0 to 12.0 at 37℃. The biochemical reaction of the substance was positive to Anthrone, Molisch and Phenol-sulfuric acid, but negative to Ninhydrin, Biuret and Benedict. A strong inhibitory activity against hydrolysis by α-amylase of human saliva, Bacillus subtilis, and porcine pancreas and glucoamylase of Aspergillus niger was shown 87, 81, 79 and 63%, respectively. but it did not inhibit cellulase of Aspergillus sp. and invertase of Saccaromyces cerevisiae.

Purification, Characterization, and Biochemical Properties of α-Amylase from Potato

( Goutam Kumar Sarker ),( Sohel Hasan ),( Farjana Nikkon ),( Ashik Mosaddik ),( Niranjan Kumar Sana ),( Habibur Rahman ),( Sang Gyu Park ),( Dong Sun Lee ),( Somi Kim Cho ) 한국응용생명화학회 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.1

Purification, characterization and biochemical properties of α-amylase from post harvest Bangladeshi Potato (Solanum tuberosum L.) were investigated. The α-amylase was purified by successive chromatography on DEAE and CM-cellulose columns with a yield of 24.24%. SDS-PAGE showed a molecular weight of 44 kDa for the enzyme that contain 2.8% sugar. The enzyme lost total activity in the presence of the chelating agent EDTA, confirming it was an α-type amylase. The enzyme displayed optimum activity at pH 7.2 and 37˚C, with an apparent Km value of 0.26% using starch as its substrate. The enzyme was strongly inhibited by Cu2+, Fe2+ and Zn2+; moderately by Li+, Hg+ and Cd2+; and slightly by Ag+, K+, Mn2+ and Mg2+. Conversely, Fe3+ and Na+ appreciably enhanced activity, while adding calcium ion nearly doubled enzyme activity. In addition, the activity of α-amylase gradually decreased with increasing concentrations of urea. Thus, potato α-amylase is an attractive target for study to better understand the structure-function relationships of α-amylases.

Bacillus brevis P176-2의 형질전환에 의한 α-amylase 생산균주 선발

채기수 慶南專門大學 1996 論文集 Vol.24 No.1

Amylase–Trypsin Inhibitors in Wheat and Other Cereals as Potential Activators of the Effects of Nonceliac Gluten Sensitivity

Yolanda Reig-Otero,Jordi Man˜es,Lara Manyes 한국식품영양과학회 2018 Journal of medicinal food Vol.21 No.3

Nonceliac gluten sensitivity (NCGS) is a gluten-related gastrointestinal disorder distinct from celiac disease (CD) and gluten allergy that is not easy to diagnose due to the lack of biomarkers. It is characterized by intestinal symptoms and extraintestinal manifestations with the consumption of gluten-containing foods. In contrast to CD, NCGS patients do not present a genetic predisposition or intestinal villi atrophy. Recent studies question the proinflammatory triggering activity of α-gliadin fraction contained in wheat, since it has been demonstrated that the amylase–trypsin inhibitors (ATIs) exert a strong activating effect on the innate immune response. We aimed to analyze the role of ATIs in the activation of innate immunity and in the development of the symptoms characteristic of NCGS. A systematic literature search was made using databases such as MEDLINE, SciELO, Science Direct, and Scopus, with focus on key words such as “amylase–trypsin inhibitors,” “wheat,” “gluten,” and “celiac.” Many studies are available on the structure, inhibition mechanism, and immune system effects of ATIs, mainly focused on IgE-mediated reactions. Recently, with the increase of NCGS interest, has increased the literature on the capacity of ATIs contained in wheat to activate the innate immune system. Literature published to date questions the relationship between activation of the innate immune system and gluten in NCGS. ATIs may have acted as interfering contaminant of gluten and appear as potential activator of innate immunity in NCGS patients. In view of their potential impact, more interventional studies are needed to demonstrate the proinflammatory effect of ATIs.

유전자조작, 균주분리 대한민국 대천 해안에서 분리한 전분 분해능을 갖는 Pseudoalteromonas sp. A-3 균주의 특징 및 동정

지원재 ( Won Jae Chi ),박다연 ( Da Yeon Park ),정성철 ( Sung Cheol Jeong ),장용근 ( Yong Keun Chang ),홍순광 ( Soon Kwang Hong ) 한국미생물생명공학회 2011 한국미생물·생명공학회지 Vol.39 No.4

Strain A-3, an amylase-producing bacteria, was isolated from coastal seawater near Daecheon in the Republic of Korea. It was seen to possess a single polar flagella and grow well, on ASW-YP agar plates, at temperatures of between 20-37℃. However, it grew more slowly at the temperatures of 15℃ and 40℃. Similarly, it was observed to grow abundantly, in an Artificial Sea Water-Yeast extract-Peptone (ASW-YP) liquid medium, in a pH range of 6-9, but not grow at pHs of 4-5 and a pH of 10. Strain A-3 was noted as being close to Pseudoalteromonas phenolica O-BC30T, Pseudoalteromonas luteoviolacea NCIMB1893T, Pseudoalteromonas rubra ATCC29570T, and Pseudoalteromonas byunsanensis FR1199T, with 98.30%, 97.86%, 97.78%, and 97.25% similarities respectively, in its 16S rRNA sequence. A phylogenetic tree revealed that strain A-3 and P. phenolica O-BC30T belong to a clade. However, strain A-3 differed from P. phenolica O-BC30T in relation to a number of physiological characteristics. Strain A-3 exhibited no growth above 5% NaCl concentrations, no utilization of D-glucose, D-mannose, D-maltose, or D-melibose, and no lipase (C-14) activity. All of these properties strongly indicate that strain A-3 is distant from P. phenolica O-BC30T and thus led us to name it Pseudoalteromonas sp. A-3. Pseudoalteromonas sp. A-3 produces α-amylase throughout growth. Maximal amylase activities of 144.48 U/mL and 149.20 U/mL were seen at pH 7.0 and 37℃, respectively. Pseudoalteromonas sp. A-3`s high, stable production of α-amylase in addition to its biochemical features, such as alkalitolerance, suggest that it is a good candidate for industrial applications.

Molecular Cloning and Characterization of a Thermostable α-Amylase Exhibiting an Unusually High Activity

박종태,AntoniusSuwanto,Irawan Tan,Tommy Nuryanto,Rudy Lukman,Kan Wang,Jay-lin Jane 한국식품과학회 2014 Food Science and Biotechnology Vol.23 No.1

An α-amylase gene was cloned from thethermophilic bacterium Bacillus subtilis isolated fromIndonesian oil palm shell waste. The gene expressed anextracellular enzyme. Optimal hydrolysis conditions for theenzyme were 70oC and pH 6.0. The specific activity of theenzyme was 16.0 kU per mg of protein, which was higherthan for other thermostable amylases. Hydrolytic productsof the enzyme using starch and glycogen were mainlymaltohexaose and maltopentaose. The enzyme had a Kmvalue of 0.099 mg/mL for amylopectin, more than 10 timeslower than for amylose. The catalytic efficiency of theenzyme using amylopectin was 39,200 mL/mg·s and was3,270 mL/mg·s using amylose. The enzyme liquefied cornstarch at pH 5.0, which was successfully converted toglucose using commercial glucoamylase and pullulanasewithout pH adjustment. The enzyme has advantages forindustrial applications.

더덕 분말 첨가량을 달리한 고추장의 효소력 변화 및 관능적 특성

성정민(Jung-Min Sung),김옥선(Ok-Sun Kim),류혜숙(Hye-Sook Ryu) 한국식품영양과학회 2011 한국식품영양과학회지 Vol.40 No.8

본 연구는 맛과 기능성을 향상시키기 위해 더덕 분말 첨가량을 달리하여 고추장을 제조하여 숙성 중 효소활성의 변화 및 관능평가를 실시하였다. 더덕분말 함량은 1%, 3%와 5%로 달리 첨가하여 30℃에 보관하면서 2주 간격으로 pH, 미생물, 유리당, 효소활성 및 관능 등의 품질을 평가하였다. 초기 pH는 더덕 첨가구가 대조구에 비해 낮았으며 저장기간 동안 감소하였다. 총균수는 저장기간 동안 큰 변화를 나타내지 않았으며 효모 및 곰팡이균은 증가하였다. 대조구와 1% 첨가구의 효모 및 곰팡이균수는 저장 6주째 5 log scale에 도달한 반면 3%, 5% 첨가구는 저장 8주째에 같은 수준에 도달하였다. 전분분해 효소인 amylase는 저장 4주째 활성이 가장 높았으며 glucose와 fructose 함량도 4주째 가장 높게 나타났다. 유리당 함량 결과 glucose와 fructose가 대부분을 차지하였으며 glucose 함량은 저장 2주째 급격하게 증가하였고 4주째 최고치에 달했으며 대조구에 비해 더덕분말 첨가 고추장의 유리당 함량이 유의적으로 높게 나타났다. 관능평가 결과 맛 및 종합적인 기호도는 1, 3% 첨가구가 대조구에 비해 높게 나타났으며 첨가구들 가운데 1% 첨가구를 가장 선호하는 것으로 나타났다. Deoduk (Codonopsis lanceolata) root powder was added to traditional kochujang to improve the quality of traditional kochujang. The microbial characteristics, enzyme activities, and sensory characteristics were investigated during fermentation. The aerobic bacterial count in kochujang was not remarkabley different, and yeast and mold decreased during fermentation. Yeast and mold in the kochujang with 3 and 5% added deoduk root powder were 5 log scale at 8 weeks, whereas those in the control reached the same level at 6 weeks. α, β-Amylase activity was at its highest level at 4 weeks during fermentation, and glucose and fructose contents showed the same results. The major free sugars in kochujang were glucose and fructose, and their contents increased rapidly at 2 weeks. Free sugar contents of kochujang with added deoduk was higher than that in the control. The sensory evaluation results showed that 1 and 3% deoduk kochujang had higher scores for taste and overall acceptance than those in the control. In particular, 1% deoduk kochujang had the highest scores.

Bacillus sp. SUH4-2로부터 생산되는 말토올리고당 생성 α-Amylase의 정제 및 특성

윤상현,김묘정,김정완,권기성,이인원,박관화 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.5

토양에서 분리된 내열성 균주인 Bacillus sp. SUH 4-2가 생산하는 maltose, maltotriose 생성 amylase를 (NH_4)_2SO_4 침전 분획, DEAE-Toyopearl 및 Mono-Q HR 5/5 column 크로마토그래피로 16.1배 정제하였으며 수율은 13.5%이었다. 이 효소의 최적 반응온도는 60∼65℃이며 60℃에서 40분간 방치된 후에도 50% 이상의 역가가 유지되었다. 최적 반응 pH는 5.0∼6.0이며 pH 5.0∼8.0에서 안정하였다. 이 효소의 등전점은 5.8이었으며 SDS-PAGE로 분석한 결과 분자량은 약 63.6 kD이었다. 이 효소를 가용성 전분과 반응시켜 생성된 당류를 TLC로 조사하여 maltose와 maltotriose가 선택적으로 많이 생성되는 것을 확인할 수 있었으며 HPIC로 정량하여 당들이 glucose : maltose : maltotriose : maltotetraose=11 : 59 : 25 : 5의 비율로 생성됨을 알 수 있었다. A Bacillus strain capable of producing an extracellular malto-oligosaccharides forming α-amylase was isolated from soil and designated as Bacillus sp. SUH4-2. The enzyme was purified by ammonium sulfate fractionation, DEAE-Toyopearl and Mono-Q HR 5/5 column chromatographies using a FPLC system. The specific activity of the enzyme was increased by 16.1-fold and the yield was 13.5%. The optimum temperature for the activity of α-amylase was 60∼65℃ and more than 50% of initial activity was retained after the enzyme was incubated at 60℃ for 40 min. The enzyme was stable over a broad pH range of 5.0∼8.0 and the optimum pH was 5.0∼6.0. The molecular weight of the enzyme was determined to be about 63.6 kD and isoelectric point was around 5.8. The enzyme activity was strongly inhibited by Mn^(2+), Ni^(2+), and Cu^(2+); slightly by Ca^(2+). The purified enzyme produced starch hydrolyzates containing mainly maltose and malto- triose from soluble starch. The starch hydrolyzates were composed of 11% glucose, 59% maltose, 25% maltotriose and 5% maltotetraose.

호염성 Bacillus s p.가 생성하는 α -amylase 특성

조용운,조수정,홍수영,김홍출 진주산업대학교 2001 산업과학기술연구소보 Vol.- No.8

양질의 간장을 제조하기 위한 목적의 일환으로서 재래식 간장으로부터 α -amylase를 분비하는 미생물을 분리하기 위하여 Soluble starch를 넣어 GPY배지를 이용하여 투명환을 크게 나타내는 균주들을 분리 선정하였다. 1. 분리균 중에서 전분 분해능이 가장 우수한 것을 선정하여 각종 생리 및 생화학적 검사, BiologTM system을 통해 이 균주가 Bacillus sp.임을 확인하고 Bacillus sp.라고 명명하였다. 2. 이 균의 생육 최적 온도는 30℃이었으며 5% NaCl 첨가 배지에서 가장 생육도가 높은 호염성균이었다. 3. 균의 크기는 0.8∼1.0×2.5∼3㎛의 단간균으로서 flagella가 있다. 4. α -amylase의 활성은 균체내 효소인 경우 pH5.4, 50℃에서 최대 활성도를 나타내었으며 균체외 효소인 경우는 pH 6, 60℃에서 최대 활성을 나타내었다. 5. Starch-SDS-PAGE 방법에 의하여 α -amylase로 추정되는 3개의 활성 밴드를 확인하였는데 분자량은 각각 97KD 이상, 66KD, 55KD이었다. A bacterial strain which produced α -amylase was isolated from soy- souce, identified to B acillus sp. by physiological and biochemical analysis. For efficient selection of α -amylase producers, microorganisms which used it in the presense of soluble starch were isolated. The optimum culture condition of B acillus sp. was 30℃ and pH7. The strain grew at the medium include 2∼10% concentration of NaCl. The α -amylase activity of the extracellular and intracellular enzyme were shown the highest level at pH 6, 60℃ and pH 5.4, 50℃. The molecular weight of α -amylase was estimated to be over 97KD, 66KD and 55KD by Starch-SDS-PAGE.

무의 α-Amylase 활성 및 가공 안정성

조은혜(Eunhye Cho),최아름(A-Reum Choi),최선주(Sun Ju Choi),김소영(Soyoung Kim),이건순(Gun Soon Lee),이수성(Soo Seoug Lee),채희정(Hee Jeong Chae) 한국식품영양과학회 2009 한국식품영양과학회지 Vol.38 No.6

소화 촉진 작용이 있다고 알려진 무의 건조방법, 온도, pH 등의 가공조건이 α-amylase 활성에 미치는 영향을 검토하였다. 동결건조 한 무 뿌리와 무 줄기의 α-amylase의 활성을 비교한 결과 무 뿌리가 무 줄기의 3.1배 높은 수준의 활성을 보였다. 무를 pH와 온도를 달리하여 안정성을 측정한 결과, pH 범위가 4~7이며 온도가 25~40℃일 때 α-amylase 활성이 높았다. 무의 α-amylase는 산성이나 중성 조건에서 처리하고 60℃ 이하의 온도로 가열처리하여야 활성을 유지하였다. 무를 깍두기와 단무지의 형태로 가공하였을 때 무의 α-amylase 잔류활성은 각각 15.39%와 19.193%이었고 초절임무에서는 α-amylase의 활성이 대부분 소실됨을 보였다. 결과적으로, 무의 소화효소인 α-amylase 활성은 열과 pH 등의 가공조건에 의해 크게 영향을 받는 것을 알 수 있었으며 60℃ 이하의 온도에서 열처리하는 것과 중성이나 약산성에서 α-amylase 효소의 활성도가 유지되는 것으로 판단된다. The effects of processing conditions on the α-amylase activity of radish were investigated at various temperatures, pHs and drying conditions. The α-amylase activity of radish root was 3.1-fold higher than that of radish trunk. As the freeze-dried radish was incubated at various temperatures and pHs, α-amylase activity was stably maintained at pH range of 4~7 and temperature of 25~40℃. When radish was processed to kakdugi and danmooji, the residual α-amylase activity was 45.39% and 19.19%, respectively. Consequently, the α-amylase activity was greatly affected by processing conditions such as heat treatment and pH. It is suggested that radish should be processed at below 60℃ and at neutral to acidic pH condition.