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研究生: 施麗英
Nani - Wibowo
論文名稱: e-Poly-L-Lysine Purification Using CMC-Magnetite and Its Application
e-Poly-L-Lysine Purification Using CMC-Magnetite and Its Application
指導教授: 李振綱
Cheng-Kang Lee
口試委員: 劉懷勝
none
王孟菊
none
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2007
畢業學年度: 95
語文別: 英文
論文頁數: 80
中文關鍵詞: e-poly-L-lysinepurificationmagnetite
外文關鍵詞: e-poly-L-lysine, purification, magnetite
相關次數: 點閱:253下載:6
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-Poly-L-lysine (-PL) is a homo-poly-amino acid characterized by the peptide bond between carboxyl and -amino groups of L-lysine. -PL is produced by several strains of Streptomyces, typically Streptomyces albulus as an extracellular product. -PL shows a wide antimicrobial activity including Gram positive, Gram negative, yeast and fungi. The minimum inhibitory concentration of -PL was measured based on broth dilution method. 62.5 mg/L of -PL was required to inhibit the growth of E. coli and B. subtilis, while 500 mg/L of -PL was required to inhibit the growth of P. pastoris.
CMC-magnetite was synthesized by mixing CMC with iron mixture solution in alkaline condition. As much as 0.0125 equivalence of carboxylic acid group was contained for each gram of CMC-magnetite. Having the diameter of 30 nm and zeta potential of -15.60 mV at pH 7, CMC-magnetite was employed to purify -PL through electrostatic interaction with the maximum capacity of 0.3788 g -PL/g CMC-magnetite. The dissociation constant of the adsorption isotherm was 0.3770 g/L and 60.77% of adsorbed -PL could be recovered from the CMC-magnetite.
-PL was employed on biomedical application. It was immobilized on microbial cellulose. 5% of NaIO4 with 2 h incubation was required to activate the microbial cellulose. As much as 5 mg of -PL was immobilized on each gram of microbial cellulose under alkaline condition. -PL-modified microbial cellulose was positive on ninhydrin assay and contained 56.06 mM amino group based on glycine calibration curve.


-Poly-L-lysine (-PL) is a homo-poly-amino acid characterized by the peptide bond between carboxyl and -amino groups of L-lysine. -PL is produced by several strains of Streptomyces, typically Streptomyces albulus as an extracellular product. -PL shows a wide antimicrobial activity including Gram positive, Gram negative, yeast and fungi. The minimum inhibitory concentration of -PL was measured based on broth dilution method. 62.5 mg/L of -PL was required to inhibit the growth of E. coli and B. subtilis, while 500 mg/L of -PL was required to inhibit the growth of P. pastoris.
CMC-magnetite was synthesized by mixing CMC with iron mixture solution in alkaline condition. As much as 0.0125 equivalence of carboxylic acid group was contained for each gram of CMC-magnetite. Having the diameter of 30 nm and zeta potential of -15.60 mV at pH 7, CMC-magnetite was employed to purify -PL through electrostatic interaction with the maximum capacity of 0.3788 g -PL/g CMC-magnetite. The dissociation constant of the adsorption isotherm was 0.3770 g/L and 60.77% of adsorbed -PL could be recovered from the CMC-magnetite.
-PL was employed on biomedical application. It was immobilized on microbial cellulose. 5% of NaIO4 with 2 h incubation was required to activate the microbial cellulose. As much as 5 mg of -PL was immobilized on each gram of microbial cellulose under alkaline condition. -PL-modified microbial cellulose was positive on ninhydrin assay and contained 56.06 mM amino group based on glycine calibration curve.

ABSTRACT ………………………………………………………………………… i ACKNOWLEDGMENT ……………………………………………………………. ii TABLE OF CONTENTS …………………………………………………………… iii LIST OF FIGURES …………………………………………………………………. vi LIST OF TABLES ………………………………………………………………….. viii CHAPTER I INTRODUCTION ………………………………………………….. 1 CHAPTER II MICROBIAL SCREENING, PRODUCTION AND ASSAY ……… 4 2.1 Literature Study ………………………………………………. 4 2.1.1 Physicochemical properties …………………………… 4 2.1.2 Antimicrobial activity ………………………………… 4 2.1.3 Microbial screening …………………………………… 7 2.1.4 Production …………………………………………….. 8 2.1.5 Quantitative assay …………………………………….. 10 2.2 Objectives …………………………………………………….. 11 2.3 Experiment Procedures 2.3.1 Microbial strain ……………………………………….. 11 2.3.2 Experiment culture medium …………………………... 11 2.3.3 Experiment reagent …………………………………… 12 2.3.4 Experiment apparatus …………………………………. 12 2.3.5 Experiment steps ……………………………………… 13 2.3.5.1 Microbial screening of -PL producer ……… 13 2.3.5.2 -PL production …………………………….. 13 2.3.5.3 -PL assay method …………………………. 13 2.3.5.4 Minimum inhibitory concentration Determination ……………………………… 18 2.4 Results and Discussion ………………………………………… 18 2.4.1 Polylysine bioassay plate ……………………………... 18 2.4.2 Blue plate assay ……………………………………….. 21 2.4.3 Polylysine PAGE ……………………………………… 23 2.4.4 Minimum inhibitory concentration ……………………. 27 CHAPTER III -POLY-L-LYSINE PURIFICATION USING CMC-MAGNETITE 30 3.1 Literature Study ……………………………………………….. 30 3.1.1 -PL purification ………………………………………. 30 3.1.2 Biomolecules purification using magnetic particle …… 30 3.1.3 CMC-magnetite synthesis …………………………….. 31 3.2 Objectives …………………………………………………….. 32 3.3 Experiment Procedures ……………………………………….. 33 3.3.1 Experiment reagent …………………………………… 33 3.3.2 Experiment apparatus …………………………………. 35 3.3.3 Experiment steps ……………………………………… 35 3.3.3.1 CMC-magnetite preparation and characterization ……………………………... 35 3.3.3.2 Optimum purification condition …………….. 36 3.4 Results and Discussions ………………………………………. 39 3.4.1 CMC-magnetite preparation and characterization ……. 39 3.4.2 Optimum purification condition ……………………… 42 CHAPTER IV APPLICATION AS WOUND DRESSING ………………………… 51 4.1 Literature Study ……………………………………………….. 51 4.1.1 Introduction to modern wound dressing ………………. 51 4.1.2 Introduction to microbial cellulose ……………………. 53 4.2 Objectives ……………………………………………………… 56 4.3 Experiment Procedures ………………………………………… 56 4.3.1 Experiment reagent …………………………………….. 56 4.3.2 Experiment apparatus ………………………………….. 57 4.3.3 Experiment steps ………………………………………. 57 4.4 Results and Discussions CHAPTER V CONCLUSION 5.1 Microbial Screening, Production and Assay ………………….. 65 5.2 -Poly-L-Lysine Purification using CMC-Magnetite ………… 65 5.3 Application as Wound Dressing ……………………………… 66 REFERENCES ……………………………………………………………………… ix

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