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«ISOLATION, EXPRESSION AND CHARACTERIZATION OF AN -L-ARABINOFURANOSIDASE ENZYME FROM Thermophilic Geobacillus sp. A Thesis Submitted to the Graduate ...»

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ISOLATION, EXPRESSION AND

CHARACTERIZATION OF AN

-L-ARABINOFURANOSIDASE ENZYME

FROM Thermophilic Geobacillus sp.

A Thesis Submitted to

the Graduate School of Engineering and Sciences of

zmir Institute of Technology

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

in Chemistry by Hüseyin LGÜ July 2011 ZM R i We approve the thesis of Hüseyin LGÜ ________________________________

Assist. Prof. Dr. Gül ah ANLI-MOHAMED Supervisor _______________________________

Assoc. Prof. Dr. Talat YALÇIN Committee Member _______________________________

Assoc. Prof. Dr. Ahmet KOÇ Committee Member 06 July 2011 _____________________________ __________________________

Prof. Dr. Serdar ÖZÇEL K Prof. Dr. Durmu Ali DEM R Head of the Department of Chemistry Dean of the Graduate School of Engineering and Sciences ii

ACKNOWLEDGEMENTS

I am heartily thankful to my supervisor Assist. Prof. Gül ah ANLI for giving me opportunity to work with her and also I would like to thank her for her encouragement, supervision, criticism and patience to guide me during my thesis studies.

I also would like to thank to Assist. Prof. Dr.Alper ARSLANO LU, Assist.

Prof. Dr. H. Ça lar KARAKAYA and Assoc. Prof. Dr. Ahmet KOÇ for their advices, confidence, help, suggestions and contributions in different parts of my thesis studies.

Throughout my thesis studies, because they all shared some instruments, materials and methods with me, I really want to thank to Molecular Microbiology, Molecular Immunology and Gene Regulation, Molecular Genetics, Molecular Bacteriology Lab, Mass Spectrometry – Proteomics Laboratory and Biotechnology and Bioengineering Central Research Laboratories members.

This thesis would not have been possible unless their supports, advices and helps during my thesis project, for this purpose, I am also thankful to my friends Yusuf SÜRMEL, Taylan TURAN, Melda Zeynep GÜRAY, Mehmet lyas CO ACAK, Gönensin Ozan BOZDA, Aylin CAMGÖZ, Ça da GÖKTA, Gözde BEKK, Cenk DA LIO LU, brahim ÇEL K, Nergiz GÜRBÜZ, Aysun ADAN, Hatice Y T, A.

Banu DEM R, rem ULUI IK, Ali Kemal HAVARE, Serdal OKUR, Merve DEM RKURT and Nadir ARAS.

I am especially grateful to LGÜ family for both mental and financial supports.

Finally, I owe my deepest gratitude to the most patient person my fiance; Ekin ÖZTÜRK. Without their endless encouragement, support and love, it wouldn`t be possible to finish this thesis.

–  –  –

In our study, we have aimed first to isolate an -L-arabinofuranosidase (ALAF) enzyme, 58.0 kDa, from a thermophilic organism; Thermophilic Geobacillus sp. by using molecular cloning techniques, then to characterize this enzyme via biochemical methods.

Throughout the characterization studies, we have investigated the optimum conditions for the highest enzyme activity by means of pH and temperature by using pNP- -L-arabinofuranoside as substrate. Also, effect of various metal ions, some specific chemicals and common organic solvents on enzyme activity was studied. Due to the fact that -L-arabinofuranosidases mainly hydrolyze -L-arabinofuranosyl residues of L-arabinose containing polysaccharides, enzyme activity towards sugar beet arabinan was also studied. Our enzyme exhibited activity in a broad pH range between pH 3.0-10.0 at 50˚C and between 30-90˚C in Na-acetate buffer pH 5.0. Optimum activity towards pNP- -L-arabinofuranoside was obtained at pH 5.0 and at 70˚C.

Kinetic studies showed that our enzyme has a Km value as 0.19 mM and Vmax as 18.6 Abs/min/ml towards pNP- -L-arabinofuranoside and Km value as 0.1 mM and Vmax as

8.1 Abs/min/ml towards sugar beet arabinan.

–  –  –

Thermophilic Geobacillus TÜRÜNDEN -L-ARAB NOFURANOZ DAZ

ENZ M N N ZOLE ED LMES, FADELENMES VE KARAKTER ZE ED LMES

Bu çalı mada, termofilik bir organizma olan Thermophilic Geobacillus türünden, moleküler klonlama teknikleri kullanılarak -L-arabinofuranozidaz ( -LAbFase) enziminin izolasyonu ve sonrasında biyokimyasal yöntemlerle karakterizasyonu amaçlanmı tır.

Karakterizasyon çalı maları sırasında, enzimin pNP- -L-arabinofuranozit substratına kar ı en yüksek verimde çalı tı ı optimum pH ve sıcaklı ı de erlerini ara tırdık. Buna ek olarak, çe itli metal iyonlarının, bazı özel kimyasallar ve yaygın kullanımı olan organik çözgenlerin enzim aktivitesi üzerine olan etkileri çalı ıldı.

Çalı tı ımız enzimin ana olarak L-arabinoze içeren polisakkaritlerde -Larabinofuranozil gruplarını parçalamasından dolayı pancar ekerinden elde edilmi olan arabinan susbtrat olarak kullanılmı tır. Enzimimiz, 50˚C sıcaklıkta geni bir pH aralı ında; pH 3.0-10.0, ve Na-asetat tamponu içinde iken 30 ile 90˚C arasında aktivite göstermi olup, pNP- -L-arabinofuranozit substratına kar ı optimum çalı ma ko ulları pH 5.0 ve 70˚C olarak saptanmı, kinetik çalı malar sonrasında enzimin bu substrata kar ı Km ve Vmax de erleri sırasıyla 14.75 mM ve 1.6 Abs/dak/ml olarak, eker pancarından elde edilmi arabinana kar ı ise sırasıyla 1.35 mM ve 3.9 Abs/dak/ml olarak hesaplanmı tır.





–  –  –

LIST OF FIGURES

LIST OF TABLES

LIST OF ABBREVIATION

CHAPTER 1. INTRODUCTION

1.1. Thermophiles

1.2. Thermophilic Enzymes

1.3. Applications of Extremozymes

1.4. Cell Wall Structure and Hemicellulose

1.5. Xylan

1.6. -L-Arabinofuranosidases

1.7. Applications of -L-Arabinofuranosidases in Industry

1.8. Aim of This Study

CHAPTER 2. MATERIALS AND METHODS

2.1. Materials

2.1.1.Chemicals

2.1.2. Medias

2.1.3. Reagents and Solutions

2.2. Methods

2.2.1. DNA Isolation and Gene Expression

2.2.1.1. Growth Condition For Bacteria

2.2.1.2. Genomic DNA Isolation

2.2.1.3. Primer Design

2.2.1.4. PCR Amplification

2.2.1.5.Agarose Gel Electrophoresis and Gel Extraction/PCR Product Purification from Agarose Gel

2.2.1.6. Protocol For Competent Cell Preparation

vi 2.2.2. Cloning the -L-AbFase Enzyme Coding Gene in Cloning Vector;

pTZ57R/T

2.2.2.1. Cloning Vector; pTZ57R/T

2.2.2.2. Transformation to DH5 Competent Cells

2.2.2.3. Plasmid Isolation

2.2.2.4. Sequence Analysis and Digestion of -L-AbFase Gene........ 14 2.2.2.5. Phylogenetic Analysis of -L-AbFase Enzyme

2.2.2.6. Expression Vector; pET28a(+)

2.2.2.6.1. Double Digestion of the Insert and Expression Vector... 15 2.2.2.6.2. Ligation of the Double Digested Insert and Vector......... 17 2.2.2.7. Transformation to BL21 ( DE3) Competent Cells................ 17 2.2.3. Protein Expression and Production

2.2.3.1. Effect of Changing Temperature on Induction of

-L-AbFase

2.2.3.2. Co-expression of -L-AbFase with Chaperon Proteins......... 18 2.2.4. Total Protein Extraction

2.2.4.1. Protein Purification

2.2.4.1.1. Affinity Chromatography

2.2.4.1.2. Size-Exclusion Chromatography

2.2.4.1.3. Dialysis

2.2.5. SDS-PAGE Analysis

2.2.6. Activity Determination of the Enzyme

2.2.6.1. Optimization of the Conditions for the Enzyme Activity......... 22 2.2.6.2. Effect of Metal Ions and Chemicals on Enzyme Activity........ 23 CHAPTER 3. RESULTS AND DISCUSSION

3.1. DNA Isolation and Gene Expression

3.1.1. Genomic DNA Isolation

3.1.2. Amplification of -L-AbFase Coding Gene

3.1.3. Cloning the -L-AbFase Coding Gene inCloning Vector;

pTZ57R/T

3.1.4. Phylogenetic Analysis of -L-AbFase Enzyme

3.1.5. Protein Expression, Production and Purification

3.2. Protein Characterization

–  –  –

CHAPTER 4. CONCLUSION

APPENDICES

APPENDIX A. CHEMICALS USED IN EXPERIMENTS

APPENDIX B. MEDIAS

APPENDIX C. REAGENTS AND SOLUTIONS

REFERENCES

–  –  –

Figure Page Figure 1.1. Potential sources of the thermophiles

Figure 1.2.

Structure of Plant Cell Wall

Figure 1.3.

(a) Structure of xylan and the sites of attack by xylanolytic enzymes. The backbone of the substrate is composed of 1,4- -linked xylose residues.

(b) Hydrolysis of xylo-oligosaccharide by -xylosidase

Figure 2.1.

pTZ57R/T cloning vector and multiple cloning sites

Figure 2.2.

pET28a (+) cloning/expression region

Figure 2.3.

pET28a (+) expression vector and multiple cloning sites

Figure 2.4.

Formation of aggregate after protein expression

Figure 2.5.

Schematic presentation of the transformation of the chaperon plasmids into BL21 competent cells contains -L-AbFase coding gene cloned with pET28a(+) vector

Figure 3.1.

Agarose gel image of PCR product

Figure 3.2.

Nucleotide sequence comparison between our protein sequence and the source gene

Figure 3.3.

Protein sequence comparison between our protein, DQ387046 and the source gene

Figure 3.4.

Phylogenetic tree of -L-AbFase

Figure 3.5.

M: Protein marker, CE: Co-expressed protein cell lysate, SE: Cell lysate of single expressed protein, P: Purified protein using Ni-affinity chromatography, W: Fraction pooled with washing buffer, FT: Flow through of Ni-affinity chromatography

Figure 3.6.

Relative activity of enzyme at different pH values

Figure 3.7.

Relative activity of enzyme at different temperature values

Figure 3.8.

Stability profile of the enzyme at different temperature values after 15 minutes

Figure 3.9.

Stability profile of the enzyme at different temperature values after 30 minutes

Figure 3.10.

Stability profile of the enzyme at different temperature values after 60 minutes

ix Figure 3.11.Stability profile of the enzyme at different temperature values after 120 minutes

Figure 3.12.

Stability profile of the enzyme at different pH values after 30 minutes... 34 Figure 3.13. Kinetic parameters against pNP- -L-arabinofuranoside

Figure 3.14.

Stability profile of the enzyme at different pH values after 120 minutes. 35 Figure 3.15.

Kinetic parameters against pNP -L-Arabinofuranoside

Figure 3.16.

Kinetic parameters against sugar beet arabinan

Figure 3.17.

Amino acid residues located around the Glu294

–  –  –

Table 1.1.

Thermophiles and their environments

Table 1.2.

Examples of extremophiles in industry and biotechnology

Table 1.3.

Properties of some microbial -L-arabinofuranosidases

Table 1.4.

Some of the potential applications of -L-arabinofuranosidases.................. 8 Table 3.1. Effect of metal ions at low concentrations on enzyme activity................... 37 Table 3.2.

Effect of metal ions at high concentrations on enzyme activity.................. 37 Table 3.3.

Relative enzyme activity after treatment with organic solvents

–  –  –

Enzymes are said to be catalysts of biological and chemical processes so, they have been used in a wide range of processes in industry and the scientists have studied on different types of enzymes to make use of new ones since the beginning of 1950s. The purpose of introducing enzymes into the industry is that not only they are said to decrease the activation energy of the reaction to take place but also they are used to eliminate the use of toxic chemicals and degrade harmful products.

1.1. Thermophiles

Environmental factors such as temperature is important for all living organisms to survive. So, according to their relation to temperature, to classify the living organisms is essential for biological systematics (Kristjansson 1989).

Based on their optimal growth temperatures, microorganisms are divided into three main groups, i.e. psychrophiles (below 20°C), mesophiles (moderate temperatures), and thermophiles (high temperatures, above 55°C) (Brock 1986), hyperthermophiles (above 80°C) (Kristjansson and Stetter 1992). Additional classification was made by Baker et al. (Baker et al., 2001) for the thermophilic organisms and in this case, they divided the thermophiles into three groups based on their minimal and maximal growth temperatures as follows: moderate thermophiles (35-70ºC), extreme thermophiles (55-85ºC) and hyperthermophiles (75-113ºC).

The most common habitats for the thermophilic organisms are geothermally and volcanically heated hydrothermal systems such as solfataric fields, neutral hot springs and submarine saline hot vents (Grant 1998), some of the bacterial and archaeal thermophiles are listed in Table 1.1 and in the Figure 1.1. some of the thermophile sources are given. So, the thermophilic organisms are able to live at high temperatures. Due to the fact that having this property, the proteins or enzymes from such organisms, generally show thermostability/activity at high temperatures.(Baker et al. 2001) Table 1.1.

Thermophiles and their environments.

(Source: Hough and Danson 1999)

–  –  –



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