고정화(固定化) Glucose Oxidase의 반복(反復) 사용성(使用性) 연구(硏究)

김성기,Kim, Sung-Kih 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.4

혈당(血糖)을 비롯한 탄수화물의 분해물 중의 글루코우즈를 측정하는데 사용되는 glucose oxidase를 고정화(固定化)시켜 반복해서 사용할 수 있는 조건을 검토하였다. GOD를 고정화 시킬 때의 가용성 GOD의 단위가 1~2일 때 그 잔여활성은 80 % 정도였고 가용성 GOD의 단위의 증가에 비례하여 잔존활성은 증가되지 않았다. 고정화 GOD의 bead의 크기가 0.3 mm로 작을 때 그 잔존활성은 85.5 %로 높았고 bead가 커질수록 그 잔존활성은 점점 저하되었다. GOD의 pH에 대한 안정성은 가용성 GOD를 pH 5.5에 10시간 처리했을 때, 60 %의 잔존활성을 보였고 고정화 GOD는 5.5~6.5에서 약 80 %정도 높은 활성을 보였다. GOD의 열에 대한 안정성은 가용성 GOD는 $35{\sim}50^{\circ}C$에서 10시간 처리해도 안정하였고 고정화 GOD는 $60^{\circ}C$에서도 안정하였으나 그 이상의 온도에서는 모두 활성이 급격히 상실되었다. 고정화 GOD를 매일 일회씩 30회 계속하여 반복 사용하여도 활성에 변화가 없었다. 고정화 GOD를 실온에서 건조하여 보관할 경우 활성이 저하되었으나 phosphate 완충액 또는 냉동건조시킨 후 $-5^{\circ}C$에 보관할 경우 큰 변화가 없었다. 고정화 GOD를 이용하여 글루코우즈는 일정하게 측정되었다. Glucose oxidase (GOD) was immobilized by polyacrylamide entrapment to use respeatedly for the determination of glucose in blood and hydrolyzates of polysaccharides. The retained activity of immobilized GOD was about 80 % when 1~2 unit of the native GOD was applied for immobilization, but the GOD activity was not retained in proportion to increment of GOD unit. The immobilized GOD had 85.5% of the ratained activity for 0.3 mm of bead diameter. The enzyme activity, however, decreased as size of of bead increased. In pH stability of the enzyme, the residual activity of the native GOD was 60% when the enzyme was pretreated at pH 5.5 for 10 hr, while the residual activity of immobilized one was about 80% when at pH 5.5~6.5. The GOD had higher thermal stability when the native enzyme was preincubated at $35{\sim}50{\circ}C$ for 10 hr and immobilized one at $35{\sim}60{\circ}C$. There was no significant decrease of the immobilized enzyme activity by using repeatedly in batch reactor one time per day for 30 times. The retained activity of immobilized GOD decrease remarkably when the product was stored at room temperature for 4 months in dry state. Lyophilized enzyme and the enzyme in phosphate buffer solution at $-5{\cirs}C$ showed a tendency to decrease depending on the storage period. The linear curve was obtained by determination of various concentrations of D-glucose by the immobilized GOD.

Purification and Amino-Terminal Amino Acid Sequence of a Ribonuclease from Bovine Seminal Plasma

김성기,Kim, Sung-Kih 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.1

소의 정액으로부터 리보뉴클리아제를 50-70% 포화 황산암모늄에 의한 침전 반응과, concanavalin A-Sepharose 4B, DEAE-셀룰로오스, agarose-5'-(4-aminophenylphos pho)-uridine 2'(3')-phosphate, Sephadex G-75 등에 의한 크로마토그라피로 정제 하였 다. 정제된 효소의 균일성은 폴리아크릴아미드 젤 전기영동으로 확인하였다. 이 효소의 분자량은 젤 여과법으로 12,500임을 알았다. 또 이 효소의 활성은 $Na^+$, $K^+$, $Mg^{2+}$, $Fe^{2+}$ 및 EDTA에 의하여 증가 되었고, $Ca^{2+}$, $Mn^{2+}$, $Zn^{2+}$ 및 $Cu^{2+}$에 의하여 억제되었다. 정제된 효소는 poly(C) 및 효모 RNA 보다는 poly(U)에 대하여 더 큰 친화성을 나타내었다. 정제된 효소 단백질 N-말단 아미노산 배열을, 아미노산 배열 자동분석기로 31번째 잔기까지 결정하였다. 그 결과는 다음과 같다. $\array{1\\Val}$ Asp Ser Lys $\array{5\\Gly}$ Gly Lys Tyr Gln $\array{10\\Arg}$ Glu His Met Asp $\array{15\\Leu}$ Asp Ser Leu Pro $\array{20\\Ala}$ Ala Val Gly Thr $\array{25\\Tyr}$Cys Asp Ala Met $\array{30\\Lys}$ Leu 이상의 결과로부터 소의 정액에서 정제한 리보뉴클리아제는 전립선-특이 효소임을 알 수 있었다. A ribonuclease has been purified to homogeneity from bovine seminal plasma by precipitation with 50-70% saturated ammonium sulfate, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose, agarose-5'-(4-aminophenylphospho)-uridine 2'(3')-phosphate, and Sephadex G-75. The homogeneity of this ribonuclease was confirmed by polyacrylamide gel electrophoresis. Molecular weight for this purified ribonuclease was 12,500 as estimated by gel filtration. The enzyme activity was activated by $Na^+$, $K^+$, $Mg^{2+}$, $Ba^{2+}$, $Fe^{2+}$ and EDTA and inhibited by $Ca^{2+}$, $Mn^{2+}$, $Zn^{2+}$, and $Cu^{2+}$ The purified ribonuclease preferentially hydrolyzed poly(U) over poly(C) and yeast RNA. The N-terminal amino acid sequence of 31 residues for the purified seminal ribonuclease has been determined by automatic sequencing of the native protein. The following sequence was deduced: $\array{1\\Val}$ Asp Ser Lys $\array{5\\Gly}$ Gly Lys Tyr Gln $\array{10\\Arg}$ Glu His Met Asp $\array{15\\Leu}$ Asp Ser Leu Pro $\array{20\\Ala$ Ala Val Gly Thr $\array{25\\Tyr}$ Cys Asp Ala Met $\array{20\\Lys}$ Leu These results suggested that the purified bovine seminal ribonuclease was a prostate-specific enzyme.

소 정액 리보뉴클리아제의 정제 및 아미노 - 말단구조에 관한 연구

김성기 ( Sung Kih Kim ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.1

A ribonuclease has been purified to homogeneity from bovine seminal plasma by precipitation with 50-70% saturated ammonium sulfate, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose, agarose-5`-(4-aminophenylphospho)-uridine 2`(3`)-phosphate, and Sephadex G-75. The homogeneity of this ribonuclease was confirmed by polyacrylamide gel electrophoresis. Molecular weight for this purified ribonuclease was 12,500 as estimated by gel filtration. The enzyme activity was activated by Na^+, K^+, Mg^(2+), Ba^(2+), Fe^(2+), and EDTA and inhibited by Ca^(2+), Mn^(2+), Zn^(2+), and Cu^(2+). The purified ribonuclease preferentially hydrolyzed poly(U) over poly(C) and yeast RNA. The N-terminal amino acid sequence of 31 residues for the purified seminal ribonuclease has been determined by automatic sequencing of the native protein. The following sequence was deduced: Val^1 Asp Ser Lys Gly^5 Gly Lys Tyr Gln Arg^(10) Glu His Met Asp Leu^(15) Asp Ser Leu Pro Ala^(20) Ala Bil Val Gly Thr Tyr^(25) Cys Asp Ala Met Lys Leu These results suggested that the purified bovine seminal ribonuclease was a prostate-specific enzyme.

放射線 및 化學藥品 兼用處理에 의한 감자의 貯藏硏究

金成器(Sung-Kih Kim),朴魯豊(Nou Pung Park) 한국식품과학회 1975 한국식품과학회지 Vol.7 No.3

고정화 Glucose Oxidase 의 반복 사용성 연구

김성기 ( Sung Kih Kim ) 생화학분자생물학회 1981 BMB Reports Vol.14 No.4

Glucose oxidase (GOD) was immobilized by polyacrylamide entrapment to use respeatedly fir the determination of glucose in blood and hydrolyzates of polysaccharides. The retained activity cf immobilized GOD was about 80 % when 1∼2 unit cf the native GOD was applied for immobilization, but the GOD activity was not retained in proportion to increment cf GOD unit. The immobilized GOD had 85.5% of the ratained activity for 0.3 ㎜ of bead diameter. The enzyme activity, however, decreased as size of of bead increased. In pH stability of the enzyme, the residual activity of the native GOD was 60% when the enzyme was pretreated at pH 5.5 for 10 hr, while the residual activity of immobilized one was about 80% when at pH 5.5∼6.5. The GOD had higher thermal stability when the native enzyme was preincubated at 35∼50℃ for 10 hr and immobilized one at 35∼60℃. There was no significant decrease of the immobilized enzyme activity by using repeatedly in batch reactor one time per day for 30 times. The retained activity of immobilized GOD decrease remarkably when the product was stored at room temperature for 4 months in dry state. Lyophilized enzyme and the enzyme in phosphate buffer solution at -5℃ showed a tendency to decrease depending on the storage period. The linear curve was obtained by determination of various concentrations of D-glucose by the immobilized GOD.

효소 및 미생물의 고정화에 관한 연구 (제1보) 방사선조사에 의한 Glucose Oxidase 의 고정화법

김성기 ( Sung - Kih Kim ) 한국미생물생명공학회 1979 한국미생물·생명공학회지 Vol.7 No.1

Immobioilized Biocatalysts를 이용한 環境性 廢棄物質 抑制에 관한 연구

金成器(Sung Kih Kim) 한국환경보건학회 1991 한국환경보건학회지 Vol.17 No.1

Saccharomyces cerevisiae was immobilized by incu벼ting iron oxides with ca1cium alginate, and by polyacrylamide entrapment to use repeatedly for the conversion of glucose to ethanol. Magnetic and non-magnetic immobilized yeast and polyacrylamide immobilized yeast were compared with the native yeast a batch-fermentation of ethanol from glucose. Three kinds of immobilized yeast tended almost identically, having ethanol productivity as well as the fina1 yield about the same to what was found for the native yeast. The long-term 0야rationa1 stability of three kinds of immobilized yeast were significant difference according as immobilized yeast activation or non-activation before ethanol fermentation. 1n the non-activation they lost their activity of fermentation rapidly in the beginning stage an slower at a later stage. On the other hand, in the activation with nutrient media, their activities were increased to some extent and stable in the later stage. The cell count of three kinds of immobilized yeast after activiation by incubating nutrient media, increased by a factor of about 45 to 48, whereas the fermenting capacity increased by a factor of 174 to 178. 111 the prearation of immobilized biocatalysts, magnetic matter does not seem to havely adverse affect on the properties of the microorganism. The immobilized biocatalysts by utilizing magnetic matter have some advantages, especia1ly in application of viscous media or insoluble particle-containing media, for this work was linked with microbial utilization of environmental wastes and elimination of envirnmental pollutant.

석류 과일 껍질을 활용하는 새로운 기능성 식품의 최근 연구 동향

김성기 ( Sung-kih Kim ) 한국식품영양학회 2017 韓國食品營養學會誌 Vol.30 No.2

Functional foods are of great significance since our society is accelerating into aging. An aging society has many physiological metabolic diseases such as hypertension, diabetes, heart disease, cancer, dementia and geriatric diseases. Fundamental treatments for the elderly are almost impossible and the social burden is heavy. If these diseases can be prevented or alleviated by improving dietary habits using functional foods, the significance would be very large. Pomegranate has been found to have 124 different kinds of phytochemicals. Polyphenols have a wide range of protective effects including various physiological metabolic diseases and cancers. It is necessary to develop functional foods such as preservatives and food extenders which can contribute to food safety, required in the food industry, by using such bioactive substances. Pomegranates have been reported to decrease the impact of many serious illnesses. There is a considerable amount of bioactive substances in the peel of a pomegranate, which has potent anticancer, antioxidant, antimicrobial and anti-apoptotic properties. Unfortunately, the peel is typically discarded after processing. Despite knowledge regarding the bioactive substances in the pomegranate peel and peel extracts, including their functionality and diversity, the knowledge is not well known by consumers in general. The aim of this study was to review up to date research trends for processing and developing new functional foods by utilizing nutritional functional substances, favourite food materials, and materials for processing food contained in pomegranate peels and pomegranate peel extracts. This study will summarize the data found in pomegranate peel and pomegranate peel extract literature mainly recently published in Science Direct. There are polyphenolic compounds (ellagitannins, punicalagin, proanthocyanidin, flavonoids, polysaccharides, etc.) in the fruit peel, making up about 50% of the pomegranate’s weight. The polyphenol content of a pomegranate fruit peel is 149.91 mg/g, which is about 100 times higher than the juice. Paying attention to the fact that the ellagitannin content (14.22 mg/g) in the fruit peel is also twice as high as that of the fruit juice and seeds, that confirms the possibility of utilizing the peel as a food ingredient capable of developing new, functional bioactive foods.

소 팹시노젠의 생합성

윤주억,김성기 ( Joo Ok Yoon,Sung Kih Kim ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.3

Poly(A)-containing mRNA of pepsinogen was isolated from total RNA of bovine gastric mucosa by oligo (dT)-cellulose chromatography and Sepharose 4B chromatography, and was translated in a wheat germ cell-free system. The translation products. were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. A major translation product was pepsinogen which was synthesized in two different molecular forms with molecular weights of 45,000 and 43,000 daltons. The pepsinogen translated in the in vitro translation system had no autocatalytic activity. The peptidemaps of the translated pepsinogen which was digested by chymotrypsin and Staphylococcus aureus V8 protease were nearly identical with the corresponding peptide masps of standard bovine pepsinogen.

Complete Amino Acid Sequence of Pheasant Egg White Lysozyme-1

정혜경,김성기,윤주억,Chung, Hye-Kyoung,Kim, Sung-Kih,Yoon, Joo-Ok 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.2

꿩 난백에서 정제한 라이소자임-1의 전 아미노산 배열은 스태필로코쿠스 프로테아제와 트립신 분해로 얻어진 팹티드에 대한 자동 및 수동식 Edman 분해로서 결정하였다. 꿩 난백중에는 3종의 라이소자임이 존재하였으나, 본 연구에서는 그 중의 라이소자임 1만의 구조를 결정하였다. 꿩 난백 라이소자임-1은 129개의 아미노산을 갖는 단일 폴리펩티드였다. 꿩 난백 라이소자임의 아미노산 배열을 닭의 것과 비교한 결과 17군데가 서로 바뀐 것으로 나타났다. 즉, 닭 난백 라이소자임의 Ala 10, Gly 16, Ala 32, Lys 33, Phe 34, Asn 37, Asp 46, Asn 48, Arg 68, Arg 73, Leu 75, Asn 77, Ala 82, Ser 85, Ser 91, Asn 93 및 Lys 96 이 꿩 난백 라이소자임에서는 Val 10, Ser 16, Val 32, Asn33, Trp 34, Gly 37, Asn 46, Asp 48, Lys 68, Lys 73, Phe 75, Ala 77, Leu 82, Asn 85, Ala 91, Arg 93 및 Tyr 96으로 각각 바뀌어있다. 따라서 이 두 효소사이에는 구조석 유사성이 높은 것으로 생각된다. The complete amino acid sequence of lysozyme-1 purified from pheasant egg white was determined by automatic and manual Edman degradation of peptides obtained from Staphylococcal protease and trypsin digestions. Although three molecular species of lysozyme were found in the pheasant egg white, only one molecular species, lysozyme-1, was sequenced in this study. Pheasant egg white lysozyme-1 (PEL-1) consisted. of a single polypeptide chain of 129 amino acid residues. The presented sequence of PEL-1 was compared with that of hen egg white lysozyme (HEL). There were 17 unique replacements, i. e., Ala 10, Gly 16, Ala 32, Lys 33, Phe 34, Asn 37, Asp 46, Asn 48, Arg 68, Arg 73, Leu 75, Asn 77, Ala 82, Ser 85, Ser 91, Asn 93, and Lys 96 in the sequence of HEL were replaced by Val 10, Ser 16, Val 32, Asn 33, Trp 34, Gly 37, Asn 46, Asp 48, Lys 68, Lys 73, Phe 75, Ala 77, Leu 82, Asn 85, Ala 91, Arg 93, and Tyr 96 respectively, in that of PEL-1. Extensive structural homology was observed among the two enzymes.