Comparison of kinetic characteristics of free and immobilized lipase enzyme on chitosan nanoparticles

Number of pages: 116 File Format: word File Code: 31802
Year: Not Specified University Degree: Master's degree Category: Chemical - Petrochemical Engineering
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    Dissertation for receiving the master's degree "M.Sc."

    Direction: Biotechnology

    Abstract:

    Lipases are one of the most important enzymes in biological systems in various industries. The application of free lipase is not cost-effective in industries due to its shorter half-life. Enzyme immobilization on different bases increases stability, repeated use, ease of product isolation, greater reaction control, and lower cost. Enzyme immobilization is often done on organic or inorganic porous bases, but in recent years, various types of nanoparticles were used due to the high contact surface per unit volume. The aim of this study is the synthesis of chitosan nanoparticles and immobilization of lipase enzyme using covalan through glutaraldehyde on nanoparticle and comparing the activity and kinetic parameters of free and immobilized enzyme. First, the chitosan nanoparticle was synthesized in the laboratory, and then the enzyme was covalently stabilized through the Bashif reaction. And it was investigated and confirmed by scanning electron microscope (SEM), infrared spectroscopy (FT-IR) and zeta sizer methods. In the next step, the activity of free and stabilized lipase enzyme was measured by Pars Azmoun diagnostic kit and the standard method of paranitrophenyl palmitate (pNPP) and the kinetic parameters Km and Vm were determined. The results of electron microscope showed that the size of chitosan nanoparticles is less than 100 nm, and based on the results of zetasizer, the size of the particles before stabilization was in the range of 10-300 nm and after enzyme stabilization was in the range of 20-500 nm. Also, the FT-IR spectra of free and stabilized lipase have absorption peaks at 1645 cm-1 and 1646.9 cm-1, respectively, which confirmed the stabilization of lipase on nanoparticles. The optimum pH of free and stabilized lipase was 7.5. Also, free lipase at 30 °C and fixed at 40 °C showed the highest activity. Although it had a lower stability and half-life compared to the immobilized enzyme.

    According to the results of the present study, the lipase enzyme can be covalently immobilized on chitosan nanoparticles through glutaraldehyde, and the immobilized enzyme has activity and kinetic properties suitable for use in various industries.

    Key words: nanochitosan, pseudomonas lipase, immobilization, enzyme activity, properties Kinetics

    Chapter One

    Introductions and Generalities

    1-1-Statement of the problem:

    Lipase is an enzyme that hydrolyzes fat into fatty acid and alcohol. Lipase is a versatile enzyme that has many industrial applications. But the stability of lipase in different environments, like other enzymes, is too low to make it possible to use it under harsh conditions that are necessary for industrial applications. In addition, lipase is expensive, and from an economic point of view, it is not economical to use lipase in free form, and because the half-life of the free enzyme is less and the ability to use it repeatedly is limited, therefore, they use stabilized enzyme on different substrates. Due to its high solubility in water, it cannot be reused. Many researches investigated the use of stabilization techniques to overcome these limitations.

    Organic porous bases were often used to stabilize the lipase enzyme, but in recent years, various types of nanoparticles have been used due to the high contact surface and high volume of pores that facilitate the entry of enzyme molecules because it increases its activity and ability to be used repeatedly and operational stability.

    One ??of the suitable types of carriers Nano is chitosan particles, which is a type of biological polymer, therefore, in this research, the kinetic characteristics of free and stabilized lipase enzyme on chitosan nanoparticles will be investigated. Therefore, the most important goals of the research are: 1-Determining the level of stabilized lipase enzyme activity compared to free lipase enzyme.

    2- The use of chitosan nanoparticles as a suitable substrate for the stabilization of lipase enzyme instead of stabilizing it on organic porous bases that have lower efficiency compared to nano carriers.

    3- More frequent and effective use of lipase enzyme and also reducing the costs of the process.

    1-3-Research hypotheses:

    According to the intended goals for the current research and obtaining appropriate answers to the questions raised, the following hypotheses will be taken into consideration:

    1- The use of lipase enzyme stabilized on chitosan nanoparticle has more suitable kinetic properties than the free enzyme.

    2-Nano Chitosan is considered as one of the best organic carriers for enzyme stabilization.

    1-4- The concept of nanotechnology [1]:

    Nanotechnology is the study of particles on an atomic scale to control them. The main goal of most nanotechnology research is to form new compounds or make changes to existing materials. Nanotechnology is used in electronics, biology, genetics, aviation and even in energy studies. Nanotechnology refers to the production of useful materials, parts and systems by controlling them in the nanometer length scale and exploiting the properties and new phenomena obtained at that scale [6]. In other words, nanotechnology is a technology that includes all the activities to create a fine structure on the nano scale to change individual atoms and molecules, so that new materials and devices with desired properties can be made [7]. In this way, compounds are produced that are expected to play a key role in future markets. The main difference between nanotechnology and other technologies is the scale of materials and structures used in this technology. Of course, it is not only the small size that is considered, but when the size of the materials is placed in this scale, their inherent characteristics, such as color, strength, etc. also changes [8].

    Abstract:

    Lipase are one of the most important enzymes in biological systems and different industries. Use of free lipase is not cost effective because of low half-life. Enzyme immobilization on different supports leads to enhanced stability, reusability case of product separation and low cost. Lipases have been immobilized on various organic and inorganic supports, but in recent years nano particles were used because of high surface area in unit volume. The aim of present study was synthesis of nano-chitosan, immobilization of lipase and comparison of kinetic characteristic of free and immobilized lipases. After synthesis of the nano chitosan and immobilization of lipase, the nano particles before and after immobilization were confirmed by SEM, FT-IR, and ZETA-SIZER. Then, activity of free and immobilized enzyme were measured by two methods and kinetic parameters such as Km and Vmax were determined.

    The results of SEM showed that the diameter of synthesized nano chitosan was less than 100 nm. Also, ZETA-SIZER results showed that the size of the nano particles before and after immobilization are in the range of 10-300 and 20-500 nm respectively. Diagram of FT-IR for free and immobilized lipases showed infrared absorption peak at wavelength of 1645 cm-1 and 1646.9 cm-1 respectively that confirmed immobilization process. Lipase can be immobilized on nano chitosan using covalent bond by glutaraldehyde successfully. Immobilized lipase has appropriate activity and kinetic characteristic for application in different industries.

    Key words: Nano chitosan, Pseudomonas lipase, immobilization, enzyme activity, kinetic characteristics.

  • Contents & References of Comparison of kinetic characteristics of free and immobilized lipase enzyme on chitosan nanoparticles

    List:

    Abstract: . 1

    Chapter One: Preliminaries and Generalities

    1-1- Statement of the problem. 3

    1-2-Research objectives. 4

    1-3-Research hypotheses. 4

    1-4- The concept of nanotechnology. 5

    1-4-1-Nanotechnology applications. 5

    1-4-1-1- Production of industrial materials and products. 5

    1-4-1-2- Medicine and the human body. 6

    1-4-1-3- Sustainability of resources. 6

    1-4-1-4- food industry. 6

    1-5-enzymes. 8

    1-5-1-Properties of enzymes. 8

    1-5-2- Characteristics of enzymatic reactions. 11

    1-5-3- parameters affecting enzyme reactions. 14

    1-5-3-1- Temperature. 14

    1-5-3-2-pH. 14

    1-5-3-3-inhibitors. 15

    1-5-4- Enzyme reaction kinetics. 16

    1-5-5-factors affecting the speed and activity of enzymes. 17

    1-5-5-1-Effect of raw material concentration on enzyme reaction speed. 17

    1-5-6- Michaelis-Menten equation. 18

    1-5-7- Lenoir-Burke equation. 21

    1-5-8-lipase enzyme. 23

    1-5-8-1-use of lipase. 25

    1-5-8-2-lipase reaction kinetics. 27

    1-5-8-3-Methods for measuring lipase activity. 28

    1-6- enzyme stabilization. 30

    1-6-1- Choosing the right base and method to stabilize the enzyme. 31

    1-7- Chitin and chitosan. 32

    1-7-1- Chitin. 33

    1-7-2- chitosan. 34

    1-7-2-1- Applications of chitosan. 35

    1-8-Determining the characteristics of chitosan nanoparticles. 38

    Chapter Two: Literature and Research Background

    2-1-Review of enzyme studies. 41

    2-2-Review on the background of lipase enzyme. 42

    2-3-History of chitin and chitosan production. 44

    2-4-History of enzyme immobilization on nanoparticles. 45

    2-5-Previous research in this field. 46

    Chapter Three: Materials and Methods

    3-1- Introduction. 51

    3-2-Chemicals. 52

    3-3-Required means and devices. 54

    3-3-1- Laboratory containers. 54

    3-3-2-devices. 54

    3-4-Laboratory methods used in the thesis. 58

    3-4-1-synthesis of chitosan nanoparticles. 58

    3-4-2-Determination of lipase enzyme by covalent binding method based on Schiff base reaction 59

    3-4-3-Determining the properties of chitosan nanoparticles using scanning electron microscope 60

    3-4-4-Determining the size distribution of synthesized nanochitosan particles by light diffraction method using Zetasizer 61

    3-4-5-infrared spectroscopy (FT-IR). 62

    3-4-6- Total protein measurement by Bradford method. 62

    3-4-7-Measuring the amount of protein (lipase) immobilized on chitosan nanoparticles. 63

    3-4-8-measurement of lipase enzyme activity. 63

    3-4-8-1-standard curve of lipase activity. 64

    3-4-8-2-first method- measurement of lipase activity using the Pars test kit (Lipase DC quantitative diagnostic kit) in serum or plasma by photometric method. 66

    3-4-8-3-second method- measurement of lipase activity using the substrate paranitrophenyl palmitate (pNPP). 68

    3-4-9- Examining the effect of temperature on the activity of lipase enzyme in the free state and immobilized on nano chitosan 68

    3-4-10-Investigating the effect of pH on the activity of lipase enzyme in the free state and immobilized on nano chitosan 69

    3-4-11-Investigating the kinetic parameters of lipase enzyme free and immobilized on chitosan nanoparticles 69

    Chapter Four: Results

    4-1-Introduction. 71

    4-2-Scanning electron microscope (SEM) imaging. 71

    4-3-Fourier transform infrared spectroscopy (FT-IR). 72

    4-4- Size distribution of nanoparticles synthesized using zeta sizer. 74

    4-5-Pseudomonas lipase activity free and stabilized on chitosan nanoparticle. 75

    4-6-Investigating the effect of pH on the activity of free and immobilized Pseudomonas lipase enzyme on chitosan nanoparticle 76

    4-7-Comparison of the relative activity percentage of free and immobilized lipase enzyme at different pH 77

    4-8-Investigating the effect of temperature on the activity of free and immobilized Pseudomonas lipase enzyme on chitosan nanoparticle. 78

    9-4-Comparison of the percentage of relative activity of free and stabilized lipase enzyme at different temperatures 79

    4-10-Kinetic parameters of free and stabilized Pseudomonas lipase enzyme on chitosan nanoparticles 80

    Chapter Five: Discussion. 78

    9-4-Comparison of the relative activity percentage of free and stabilized lipase enzyme at different temperatures 79

    4-10-Kinetic parameters of free and stabilized lipase pseudomonas enzyme on chitosan nanoparticle 80

    Chapter five: discussion and conclusion

    Discussion. 83

    Conclusion. 87

    Problems and suggestions. 89

    Sources and sources. 90

    List of Persian sources. 91

    List of non-Persian sources. 92

    English abstract. 100

     

    Source:

     

    ]1[ Alamzadeh; Iran, 1377, enzyme processes, Sharif University of Technology Publishing Institute. Manouchehr, 1380, Biochemical Engineering, Sharif University of Technology Publishing Institute.

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Comparison of kinetic characteristics of free and immobilized lipase enzyme on chitosan nanoparticles