Purification, characterization, and corroborated applications of xylanase from Streptomyces strains utilizing agro-waste
- Issued Date
- 2013
- Abstract
- Two Streptomyces sp., CS628 and CSWu-1, recently isolated from Korean soil were identified according to various characteristics including 16S rDNA and rRNA sequencing, respectively. Both extracellular xylanases, Xyn628 and XynWu-1, were grown on various medium and purified using different chromatographic techniques and biochemically characterized. Xyn628 was purified by single step gel permeation chromatography using Sepharose CL-6B and XynWu-1 was purified by gel permeation chromatography using sephadex G-50 followed by weak anion exchange, DEAE-sepharose fast flow, chromatography. Activity recovery of Xyn628 and XynWu-1 were 33.78 % and 16 %, respectively, whereas purity fold were 5.16 and 4.87, respectively. The molecular masses of both xylanase were approximately 18.1 kDa (Xyn628) and 37 kDa (XynWu-1) as estimated by SDS-PAGE and xylan-zymography. Resulting, molecular weight of Xyn628 is the lowest among the so far reported Streptomyces xylanase. N-terminal amino acid sequences of Xyn628 and XynWu-1 were AYIKEVVSRAYM, AINVLVAAL, respectively. Both the sequences are significantly different from the reported xylanase. The xylanases showed high activity and stability in extremely alkaline conditions.
Optimum temperature and pH of Xyn628 and XynWu-1 were 60 °C and 11.0, respectively. Both the xylanases were stable in a wide pH ranging between pH 5.0 ~ 13.0 after 12 h of incubation at 4 °C. Xyn628 and XynWu-1 were stable up to 65 °C and 55 °C after 1 h incubation, respectively. XynWu-1 activity was remarkably enhanced by various metal ions, detergents and organic solvents. Especially, enhanced xylanase activity of XynWu-1 at higher molar concentration by Mn2+ ion suggested unique among Streptomyces, which might be a metalloprotein and metal ions that positively stimulated its activity may be employed as catalysts in trace amounts during application The xylanases, Xyn628 and XynWu-1, were most active on beechwood xylan with Km values of 3.1 and 1.7 mg/mL, respectively, and Vmax of 313 and 742 mmoL/min mg, respectively. This kinetic parameter therefore importantly defines the affinity of the substrate for the enzyme. Xyn628 and XynWu-1 produced xylobiose, xylotriose and xylose, xylobiose, xylotetraose as principal hydrolyzed end products from xylan, suggesting that they are endo-xylanase in nature. Importantly, scanning electron microscopy (SEM) showed Xyn628 and XynWu-1 efficiently degraded (corncobs, wheat bran etc.) agro-industrial waste materials. In summary, Due to extremely alkaline, thermostable, cellulase free, very low molecular weight, very high effectiveness with various detergents and metal ions, series of xylooligosaccharides production etc. of the purified xylanases make these enzymes attractive for various biotechnological applications, such as biobleaching in paper and pulp industries, production of xylooligosaccharides for prebiotic in food and pharmaceutical industry, potential application in biofuel and textile industries as well as waste treatment with appropriate utilization of agro-waste products.
- Alternative Title
- Streptomyces균주에서 xylanase의정제, 특성분석 및 산업적 응용
- Alternative Author(s)
- Md. Arifur Rahman
- Affiliation
- 조선대학교 대학원
- Department
- 일반대학원 약학과
- Advisor
- 유진철
- Awarded Date
- 2013-08
- Table Of Contents
- TABLE OF CONTENTS
TABLE OF CONTENTS i
LIST OF TABLES iv
LIST OF FIGURES v
ABBREVIATIONS vi
ABSTRACT 3
1. INTRODUCTION………………………………………5
1.1 Structure and composition of lignocellulosic biomass 6
1.2 Xylan: occurrence, structure and composition……… 9
1.3 Xylanolytic enzyme system…………………………… 11
1.3.1 Endoxylanase (β-1,4-D-xylanohydrolase, E.C. 3.2.1.8)……………………………………………………………12
1.4 Microorganisms and production of xylanase……… 13
1.5 Purification of xylanase………………………………… 14
1.6 Applications of xylanases……………………………… 16
1.7 Problem delineated……………………………………… 18
1.8 Objectives of the present study………………………… 19
2. MATERIALS & METHODS………………………………… 20
2.1 Materials…………………………………………………… 20
2.2 Strains Isolation and Identifications…………………… 20
2.3 Production of Xylanases………………………………… 20
2.3.1 Production of Xyn628……………………………… 20
2.3.2 Production of XynWu-1…………………………… 21
2.4 Determination of enzyme activity…………………………21
2.5 Protein determination…………………………………… 22
2.6 Purification of enzymes…………………………………… 22
2.6.1 Purification of Xyn628……………………………… 22
2.6.2 Purification of XynWu-1…………………………… 22
2.7 Determination of enzyme purity………………………… 23
2.8 Effect of pH on activity and stability of the xylanases…23
2.9 Effect of temperature on activity and stability of the xylanases …………………………………………………………24
2.10 Effect of detergents……………………………………… 24
2.11 Effect of metal ions, and other additives……………… 24
2.12 N- terminal amino acid sequences………………… 25
2.13 Substrate specificity of xylanases………………………25
2.14 Kinetic parameter of xylanases………………………… 25
2.15 Mode of hydrolysis and potential application of xylanases in xylooligosaccharides production…………… 26
3. RESULTS AND DISCUSSION…………………………… 27
3.1 Strains isolation and identification…………………… 27
3.2 Production of enzymes…………………………………… 29
3.2.1 Production of Xyn628……………………………… 29
3.2.2 Production of XynWu-1…………………………… 31
3.3 Purification of enzymes…………………………………… 32
3.3.1 Purification of Xyn628……………………………… 32
3.3.2 Purification of XynWu-1…………………………… 35
3.4 Effect of pH on activity and stability of the xylanases…37
3.5 Effect of temperature on activity and stability of the xylanases………………………………………………………… 41
3.6 Effect of detergents………………………………………… 43
3.6.1 Effect of detergents on Xyn628 activity………… 43
3.6.2 Effect of detergents on XynWu-1 activity……… 44
3.7 Effect of metal ions, and other additives……………… 46
3.7.1 Effect of metal ions, and other additives on Xyn628 activity……………………………………………………46
3.7.2 Effect of metal ions, and other additives on XynWu-1 activity…………………………………………………48
3.8. N- terminal amino acid sequences…………………… 51
3.9 Substrate specificity of xylanases……………………… 51
3.10 Kinetic parameter of xylanases…………………………52
3.11 Mode of hydrolysis and potential application of xylanases in xylooligosaccharides production………………………………………………………52
3.11.1 Mode of hydrolysis and potential application of Xyn628 in xylooligosaccharides production …………… 52
3.11.2 Mode of hydrolysis and potential application of XynWu-1 in xylooligosaccharides production …………55
4. CONCLUSIONS…………………………………………… 58
REFERENCES…………………………………………… 59
APENDIX 1: N-terminal sequences of Xyn628………67
APENDIX 2: N-terminal sequences of XynWu-1……… 72
- Degree
- Master
- Publisher
- 조선대학교 대학원
- Citation
- 라만 엠디 아리퍼. (2013). Purification, characterization, and corroborated applications of xylanase from Streptomyces strains utilizing agro-waste.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/9876
http://chosun.dcollection.net/common/orgView/200000263927
- Appears in Collections:
- General Graduate School > 3. Theses(Master)
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