Pseudomonas sp.로부터 알칼리내성 amylase의 정제 및 특성 확인

국문 초록 (Abstract)

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.

다국어 초록 (Multilingual Abstract)

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.

목차 (Table of Contents)

• Abstract
• 서론
• 재료 및 방법
• 결과 및 고찰
• 요약
• 감사의 글
• 문헌

참고문헌 (Reference)

1 Miller GL, "Use of dinitrosalicylic acid reagent for determination of reducing sugar" 31 : 426-428, 1959

2 Chi ZM, "Trehalose accumulation from starch by Saccharomycopsis fibuligera sdu" 28 : 240-245, 2001

3 Cheong KA, "Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium" 23 : 79-87, 2005

4 Bradford MM, "Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding" 72 : 248-254, 1976

5 Takasaki Y, "Purification and enzymatic properties of β-amylase and pullulanase from Bacillus cereus var. mycoides" 38 : 1023-1029, 1976

6 Shen GJ, "Purification and characterization of a novel thermostable β-amylase from Clostridium thermosulphurogenes" 254 : 838-840, 1988

7 Kim TU, "Purification and characterization of a maltotetraose-forming alkaline α-amylase from an alkalophilic Bacillus strain, GM8901" 61 : 3105-3112, 1995

8 Rao JLUM, "Purification and characterization of a hyperthermostable and high maltogenic α-amylase of an extreme thermophile Geobacillus thermoleovorans" 142 : 179-193, 2007

9 Das K, "Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular α-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India" 40 : 291-298, 2004

10 Kato K, "Pseudomonas intracellular amylase with high activity on maltodextrins and cyclodextrins" 391 : 96-108, 1975

11 Lowry OH, "Protein measurement with the folin phenol reagent" 193 : 265-275, 1951

12 Gupta R, "Microbial α-amylases: A biotechnological perspective" 38 : 1599-1616, 2003

13 Simpson CL, "Intracellular α-amylase of Streptococcus mutans" 180 : 4711-4717, 1998

14 Oishi M, "Intracellular α-amylase in Bacillus subtilis" 85 : 246-247, 1963

15 Whitehead TR, "Identification of intracellular amylase activity in Streptococcusbovis and Streptococcussalivarius" 30 : 143-148, 1995

16 Ballschmiter M, "Identification and characterization of a novel intracellular alkaline α-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8" 72 : 2206-2211, 2006

17 Bernhardsdotter ECMJ, "Enzymic properties of an alkaline chelator-resistant α-amylase from an alkaliphilic Bacillus sp. isolate L1711" 40 : 2401-2408, 2005

18 Igarashi K, "Enzymatic properties of a novel liquefying α-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences" 64 : 3282-3289, 1998

19 Oh KW, "Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli" 252 : 175-181, 2005

20 Nielsen AD, "Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus α-amylase: a calorimetric investigation" 373 : 337-343, 2003

21 Kang EJ, "Cloning, sequencing, characterization, and expression of a new α-amylase isozyme gene (amy3) from Pseudomonas sp" 23 : 811-816, 2001

22 Kim ES, "Cloning, sequencing and expression of the amylase isozyme gene from Pseudomonas sp. KFCC 10818" 18 : 169-174, 1996

23 Na HK, "Cloning and nucleotide sequence of the α-amylase gene from alkalophilic Pseudomonas sp. KFCC 10818" 6 : 203-208, 1996

24 Lim WJ, "Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8" 154 : 681-687, 2003

25 Laemmli UK, "Cleavage of structural proteins during assembly of head of bacteriophage-T4" 227 : 680-685, 1970

26 Liu B, "Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616" 39 : 805-810, 2006

27 Osman AB, "Amylase in chicken intestine and pancreas" 73B : 571-574, 1982

28 Hashim SO, "Alkaline active maltohexaose-forming α-amylase from Bacillus halodurans LBK 34" 36 : 139-146, 2005

29 Pandey A, "Advances in microbial amylases. Biotechnol" 31 : 135-152, 2000

30 Saxena RK, "A highly thermostable and alkaline amylase from a Bacillus sp. PN5" 98 : 260-265, 2007