Pseudomonas sp.로부터 알칼리내성 amylase의 정제 및 특성 확인

이정은(Jeong-Eun Lee),전덕영(Deok-Young Jhon) 한국식품과학회 2008 한국식품과학회지 Vol.40 No.1

두 개의 amylase를 호알칼리성 Pseudomonas sp. KFCC 10818의 배양액으로부터 정제하여 그 특성을 조사하였다. 정제된 효소의 분자량은 각각 50 kDa과 75 kDa이었다. 이 효소들의 최적반응온도는 각각 35℃와 40℃였으며 50 kDa의 효소는 칼슘이온에 의하여 효소활성이 두 배로 촉진되었다. 이 두 효소는 최적 pH가 6-8 부근이었으며 pH 12의 조건에서도 효소활성을 유지하는 알칼리내성을 나타냈다. Maltooligosaccharide이나 soluble starch로부터 maltose와 maltotriose를 최종 효소반응산물로 생산하였다. 두 amylase는 N-말단 아미노산 서열이 각각 QTVPKTTFV와 DTVPGNAFQ로 분석되었다. Two extracellular amylase isozymes were purified and characterized from alkalophilic Pseudomonas sp. KFCC 10818 for the production of maltooligosaccharides. The molecular weights of the homogeneous proteins were 50 kDa and 75 kDa, respectively. The 50 and 75 kDa amylases showed optimum temperatures at 35 and 40℃, respectively. The optimum pH of the enzymes ranged from pH 6-8, and the enzymes were resistant to an alkaline condition of pH 12. Via the enzymes actions, the final products from maltooligosaccharides or soluble starch were maltose and maltotriose. Calcium was a potent activator of the 50 kDa amylase. Finally, the N-terminal amino acid sequences of the 50 and 75 kDa amylases were QTVPKTTFV and DTVPGNAFQ, respectively.

Production of Maltopentaose and Biochemical Characterization of Maltopentaose-Forming Amylase

KIM, YOUNG-MIN,RUY, HWA-JA,LEE, SUN-OK,SEO, EUN-SEONG,LEE, SO-YOUNG,YOO, SUM-KYUN,CHO, DONG-LYUN,KIM, DOMAN,ATSUO KIMURA,SEIYA CHIBA,LEE, JIN-HA 한국미생물 · 생명공학회 2001 Journal of microbiology and biotechnology Vol.11 No.4

Bacillus sp. AIR-5, a strain from soil, produced an extracellular maltohentaose-forming amylase from amylose and soluble starch. This bacterium produced 8.9g/l of maltopentaose from 40g/l of soluble starch in a batch fermentation and the maltopentaose made up 90% of the maltooligosaccharides produced (from maltose to maltoheptaose). The culture supernatant was concentrated using a 30K molecular weight cut-off membrane and purified by DEAE-Cellulose and Sephadex G-150 column chromatographies. The purified protein showed one band on a native-PAGE and its molecular mass was estimated as 250kDa. The 250-kDa protein was composed of tetramers of a 63-kDa protein. The isoelectric point of the purified protein was pH 6.9, and the optimum temperature for the enzyme activity was 45℃. The enzyme was quickly inactivated above 55℃, and showed a maximum activity at pH 8.5 and over 90% stability between a pH of 6 to 10. The putative N-terminal amino acid sequence of AIR-5 amylase, ATINNGTLMQYFEWYVPNDG, showed a 96% sequence similarity with that of BLA, a general liquefying amylase.

Treatment of ramie leaf β-amylase for preliminary purification

Dang, Nguyen Dang Hai,Lee, Jin-Sil Korean Society of Food Science and Technology 2016 한국식품과학회지 Vol.48 No.6

The thermal properties of ramie leaf ${\beta}$-amylase (RBA) were examined to develop a novel process for enzyme purification. The thermostability of RBA extract prepared from ramie leaf powder was examined at various temperatures. RBA activity decreased slightly, whereas other carbohydrate-active enzymes, such as $\small{D}$-enzyme, were rapidly inactivated during 30 min incubation at $60^{\circ}C$. When the heat-treated extract was incubated with various substrates, maltose was produced exclusively as the major product, whereas the untreated crude extract produced maltose and other maltooligosaccharides. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, fewer protein bands were observed for the heat-treated extract than the untreated extract, indicating that the thermostable RBA was partially purified and other thermolabile enzymes were eliminated. Thus, the treatment of the RBA extract at $60^{\circ}C$ for 30 min resulted in 5.4-fold purification with a recovery yield of 90%.

Precepitation and purification of amylase enzyme produced by streptomyces aureofaciens 77

Ibrahim, A.N.,Ahmed, F.H.,Ibrahim, M.M.K.,Arafa, M.A.I. The Pharmaceutical Society of Korea 1990 Archives of Pharmacal Research Vol.13 No.1

Precipitation and purification of amylase secreted by Streptomyces aureofaciens 77 in liquid inorganic salts-starch medium under the optimum conditions were carried out. Ammonium sulphate fractionation was used to precipitate amylase in cell free culture filtrate. (NH/sub 4/)/sub 2/ SO/sub 4/ at a concentration of 50-70% saturation gave the highest enzyme yield. The obtained precipitates were redissolved in phosphate buffer (pH 7.0) and subjected to dialysis. The dialyzed enzyme preparation was applied to DEAE-cellulose column chromatography which resulted in an increase of purification up to 59.48 fold. A further step of purification was done by applying the obtained purified sample to Sephadex-G200 column chromatography which resulted in ann increase of purification up to 73. 92 fold. The results clearly indicated that the isolated amylase from S. aureofaciens 77 was only on type.

Isolation and Identification of Inhibitory Compounds from Morus alba cv. Kuksang on α-amylase and α-glucosidase

Moo-Young Choi(최무영),Young-Je Cho(조영제) 한국생명과학회 2015 생명과학회지 Vol.25 No.8

109종의 뽕잎 종류 중 국상 품종 뽕잎으로부터 α-amylase와 α-glucosidase 저해작용을 가지는 페놀성화합물을 분리하여 저해활성을 검토하였다. 국상 뽕잎의 물추출물에서 α-amylase와 α-glucosidase 저해 활성이 각각 93.8%와 48.7%를 나타내었다. 국상뽕잎 추출물의 페놀화합물의 함량은 물 추출물에서 9.7±0.2 mg/g, 60% ethanol 추출물에서 14.3±0.2 mg/g으로 나타났으며, 국상뽕잎 물 추출물 200 μg/ml의 농도에서 α-amylase와 α-glucosidase 저해활성이 각각 100%와 82.6%로 나타났다. 국상뽕잎으로부터 α-amylase와 α-glucosidase 저해활성을 가지는 천연물을 분리하기 위하여 국상뽕잎 물추출물을 Sephadex LH-20과 MCI-gel을 이용하여 normal phase type인 EtOH→H₂O와 reverse phase type인 H₂O→MeOH로 유기용매의 농도를 증가시키며 gradient로 용출하여 정제하였다. α-Amylase와 α-glucosidase 저해활성을 가지는 정제물은 FAB-MS, NMR과 IR spectrum을 구조 분석한 결과 quercetin으로 동정되었다. The objective of this research was to evaluate the inhibitory activities of phenolic compounds isolated from mulberry (Morus alba) leaves of 109 types against α-amylase and α-glucosidase. The inhibitory activity of the water extracts from Morus alba cv. Kuksang against α-amylase and α-glucosidase were determined as 93.8% and 48.7% respectively. The total phenolic content of extracts from Morus alba cv. Kuksang was 9.7±0.2 mg/g soluble in water and 14.3±0.2 mg/g soluble in ethanol. The inhibitory activity of the water extracts from Morus alba cv. Kuksang at 200 μg/ml phenolics concentration against α-amylase and α-glucosidase were determined as 100% and 82.6% respectively. The purification of inhibitory compounds was carried out by Sephadex LH-20 and MCI-gel CHP-20 column chromatography using a gradient elution procedure by nomal phase type (EtOH→distilled water) and reverse phase type (distilled water→MeOH). The quercetin was confirmed to be the chemical structure of the inhibitory compound against α-amylase and α-glucosidase by spectroscopic analysis of FAB-MS, NMR and IR spectrum.

Purification and Biochemical Characterization of a Detergent Stable α-amylase from Pseudomonas stutzeri AS22

Hana Maalej,Noomen Hmidet,Olfa Ghorbel-Bellaaj,Moncef Nasri 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.5

This study reports the purification and biochemical characterization of a novel maltotetraose-forming-α-amylase from Pseudomonas stutzeri AS22, designated PSA. The P. stutzeri α-amylase (PSA) was purified from the culture supernatant to homogeneity by Sepharose mono Q anion exchange chromatography, ultrafiltration and Sephadex G-100 gel filtration, with a 37.32-fold increase in specific activity, and 31% recovery. PSA showed a molecular weight of approximately 57 kDa by SDS-PAGE. The N-terminal amino acid sequence of the first 7 amino acids was DQAGKSP. This enzyme exhibited maximum activity at pH 8.0 and 55oC, performed stably over a broad range of pH 5.0 ~ 12.0, but rapidly lost activity above 50oC. Both potato starch and Ca2+ ions have a protective effect on the thermal stability of PSA. The enzyme activity was inhibited by Hg2+, Mn2+, Cd2+, Cu2+, and Co2+, and enhanced by Ba2+. PSA belonged to the EDTA-sensitive α-amylase. The purified enzyme showed high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), and oxidizing agents, such as sodium per borate and H2O2. In addition, PSA showed excellent compatibility with a wide range of commercial solid and liquid detergents at 30oC, suggesting potential application in the detergent industry. Maltotetraose was the specific end product obtained after hydrolysis of starch by the enzyme for an extended period of time, and was not further degraded.

Purification and partial characterization of α-amylase from soybean (Glycine max)

Tripathi, Pallavi,Dwevedi, Alka,Kayastha, Arvind M. Kyung Hee Oriental Medicine Research Center 2004 Oriental pharmacy and experimental medicine Vol.4 No.4

An ${\alpha}-Amylase$ was purified to apparent homogeneity from germinating soybean seeds (Glycine max). Enzyme showed high specificity for starch. ${\alpha}-Amylase$ from soybean has optimum pH at 7.6 in the pH range 4.0-10.6. At this pH, the $K_m$ of starch was 2.63 mg/ml and the $V_{max}$ was equal to 52.6 mg/ml/min protein. Optimum temperature of the enzyme was found to be $55^{\circ}C,\;Q_{10}$ equal to 1.85 and energy of activation equal to 12 kcal/mol. Additives like, EDTA reduced the activity of ${\alpha}-amylase$ whereas PMSF enhanced the activity. ${\alpha}-Amylase$ was inhibited by several heavy metal ions.

Schwanniomyces castellii CBS 2863(ATCC 26077)으로부터 α- Amylase 정제 및 특성

박종천,배석,임선영,이진종,이향,전순배 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

Schwanniomyces castellii CBS 2863의 배양 상징액으로부터 α-amylase를 정제하였다. Ultrafiltration과 gel filtration 두 단계를 통하여 정제된 α-amylase의 분자량은 약 56 KD이었고, glycoprotein이었다. 정제된 α-amylase 최적 pH와 온도는 각각 5.5와 40℃였고, pH 4.0∼7.0 범위와 40℃에서 안정하였다. 전분에 대한 K_m값과 V_max값은 각각 1.0 ㎎/㎖과 100 U/㎎ protein이었다. 그리고 아미노산 조성 분석결과 산성단백질이었고, N-말단 아미노산 서열은 Asp-Val-Ser-Ser-Ser-Ala-X-X-Thr-Arg-Ser-Glu-Ser-Ile-Tyr이었다. The extracellular α-amylase was purified to homogeneity from the culture filtrate of starch grown Sch. castellii CBS 2863. The purified enzyme was glycoprotein with a molecular weight of about 56 KDa. The pH and temperature optimum were 5.5 and 40℃, respectively. The enzyme was fairly stable up to 40℃ and at acid pH range(pH 4.0∼7.0). The apparent K_m and V_max of the enzyme toward starch was 1.0 ㎎/㎖ and 100 U/ ㎎ protein, respectively. The analysis of amino acid composition was found to be acidic protein. The amino acid sequence of N-terminal peptide consisted of Asp-Val-Ser-Ser-Ser-Ala-X-X-Thr-Arg-Ser-Glu-Ser-Ile-Tyr.

재조합 대장균으로부터 Pseudomonas sp. 아밀라제의 정제 및 특성

김은선 코리아뷰티디자인학회 2009 코리아뷰티디자인학회지 Vol.5 No.3

Schwanniomyces castellii CBS 2863(ATCC 26077)으로부터 $\alpha$-Amylase 정제 및 특성

박종천,배석,임선영,이진종,이향,전순배,Park, Jong-Chun,Bai, Suk,Lim, Suhn-Young,Lee, Jin-Jong,Lee, Hyang,Chun, Soon-Bai 한국미생물·생명공학회 1993 한국미생물·생명공학회지 Vol.21 No.6

The extracellular alpha-amylase was purified to homogenity from the culture filtrate of starch grown Sch. castellii CBS 2863. The purified enzyme was glycoprotein with a molecular weight of about 56 kDa. The pH and temperature optimum were 5.5 and 40C, respectively. The enzyme was fairly stable up to 40C and at acid pH range (pH 4.0-7.0). The apparent Km and Vmax of the enzyme toward starch was 1.0mg/ml and 100U/mg protein, respectively. The analysis of amino acid composition was found to be acidic protein. The amino acid sequence of N-terminal peptide consisted of Asp-Val-Ser-Ser-Ala-X-X-Thr-Arg-Ser-Glu-Ser-Ile-Tyr.