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Materials and Method
Materials
- Enzymes
The lipase used in this study is Amano lipase derived from pseudomonas fluoresces, purchased from Sigma-Aldrich (product code 534730-50G) and produced by Amano enzyme Inc, Japan. Microbial transglutaminase (mTGase) isolated from Streptomyces mobaraense was kindly donated by Yiming Biological Products Co. Ltd (China). Galactosidase from Kluyveomyces lactis was purchased from Sigma-Aldrich.
Other Chemicals
Polyethyleneimine (PEI) was bought from Aldrich (product code 25987-06-8, Mw 600,000). p-nitrophenyl palmitate (pNPP), the artificial substrate used for testing the activity of the lipase, was bought from Sigma (N-2752). Triton X-100 (which was used as a surfactant in the preparation of pNPP assay solution), was bought from BDH (product code 306324N). The buffer used in this study was prepared with Trizma-hydrochloride (Sigma, T-2253) and di-sodium tetraborate (BDH, product code 102674E) respectively. Dichlorocyanuric acid (DCCA) was purchased from the local market (as swimming pool bleaching agent). 100% woollen cloth without bleaching was kindly provided by AgResearch Limited (NZ). QuantiPro BCA Assay Kit (Bicinchoninic acid) protein assay kits were purchased from Sigma-Aldrich (product code QPBCA-1KT).
All aqueous solutions were prepared with Deionised (DI) water. All other chemicals used were of analytical grade.
Experimental techniques
Woollen Cloth Chlorination
Wool fibres are hydrophobic and chemical resistant, due to the aforementioned (Section 2.1) covalent bound lipid layer and sulfur-rich cortex layer on the surface [19] . Thus, as previously mentioned (Section 2.1) surface modification is necessary before immobilization. In this study, DCCA chlorination was adopted. This reagent is able to release chorine slowly and reduce the weight loss of wool, which is widely accepted in wool industry. The method described in references [30, 172, 173] was used in the current research with minor modification. In the wool industry, chlorine concentration is often quantified by owf, i.e. weight of cloth (% chlorine equivalent to the weight of the cloth used). For example, 5% owf chlorine solution means the amount of chlorine used is 5% of cloth weight. In this research, two types of chlorination processes were adopted:
Low pH process: In the first part of thesis, the procedure in reference [173] was used for wool chlorination with some modifications: Cloth was treated for 30 second to 3 minutes in a beaker containing 2.5% owf of DCCA. The solution pH was adjusted to 1.5-2.0 by using solutions of sulphuric acid and acetic acid. Then the cloth was soaked in neutralization solution containing 10 g.l-1sodium carbonate and 4 g.l –1 sodium sulfite for 30 sec, then rinse with water and dry.
High pH process: However it was found the woollen cloth could not be evenly chlorinated by the above procedure, thus the more moderate chlorination steps described in reference [30] was used later in this thesis, the detail discussion is given in Section 6.3. The chlorination steps were as follows: Bare woollen cloth was treated in 5% owf (weight of cloth) solution in 0.1 M citric acid monohydrate/0.15 M sodium hydroxide buffer, pH 4.1 for 60 minutes at room temperature (25 ° C) at a liquor /cloth ratio of 20:1. After chlorination, the cloth was treated in 5% owf Na2SO3 in Na2HPO4/NaOH (pH 8.5), liquor /cloth ratio 20:1, for 30 minutes at 50°C. Thereafter, the cloth was rinsed with DI water and soaked in 2% PEI solution at pH 8.5 until used.
Buffer selection and preparation
Several buffers were used in this project, namely Tris buffer, phosphate buffer, acetate buffer, and carbonate buffer.
The buffers used in immobilization step must be inert in the presence of aldehyde group. However it was found in the experimental work that amine-contained buffers, such as ammonia buffer and Tris buffer, are able to react with GA, as observed that the pH in GA solution kept on dropping, and solution colour turn to yellow very fast when amine- contained buffers are used. This observation corresponds to the conclusion made by M. A. Hayat [174]: “The Tris buffer, like other primary amines, appears to react with glutaraldehyde and therefore should be avoided except when no alternative is available”. Therefore in this project, 0.1 M phosphate buffer was selected as the main immobilization buffer.
However, Tris buffer is an excellent storage buffer, which can quench the excessive aldehyde groups on the immobilized support surface and protect the immobilized lipase activity. The importance of using blocking agents to remove excessive aldehyde group on immobilization support have been addressed in review section 2.3.2.2 clearly. The optimal pH for lipase from pseudomonas fluorescence was found in the experimental work to be 8.5 (see the results in Section 5.1.1), thus this value was selected for the storage buffer.
1. Introduction
1.1 Why use Enzymes? Advantages and Disadvantages
1.2 Objectives of this Research
1.3 Outline of the Thesis
2. Literature Review
2.1 Wool
2.2 Enzymes: Reactions and Stability
2.3 Enzyme Immobilization
2.4 Important Chemicals used for Enzyme Immobilization
2.5 Immobilization Supports
2.6 Modelling Enzyme Reactions: Enzyme Kinetics
2.7 Summary of Literature Review
3. Materials and Methods
3.1 Materials
3.2 Experimental techniques
4. Lipase and Galactosidase Immobilization by Covalent Binding Method (CVB)
4.1 Immobilization via physical Adsorption on bare Woollen Cloth
4.2 The Effect of Chlorination on the Woo
4.3 Effects of different parameters on immobilization activity
4.4 Protein Load on Immobilized Support
4.5 ESEM Images of Immobilized Lipase
4.6 Illustration of CVB Immobilization Mechanism
4.7 Immobilization of β-galactosidase via the CVB Protocol
4.8 Conclusions
5. Characterising the Immobilized Lipase
5.1 Change in Activity of the Lipase with pH
5.2 Storage Stability of free and immobilized Lipase
5.3 The oily stain removal capacity of the immobilized lipase
5.4 Kinetic Model for Free and Immobilized Lipase
5.5 Conclusions
6. Improvement of CVB Protocol for Lipase Immobilization
6.1 CVB protocol variations: Lipase Immobilization by Cross-linking Methods (CRL)
6.2 CVB protocol variation: GA Oligomer
6.3 Variation of Chlorination Method for Immobilization
6.4 CVB protocol variation: PEI-Polyaldehyde
6.5 Comparison of the immobilization protocols studied in this and previous chapters
6.6 Conclusions
7. Bi–enzyme System for Enzyme Immobilization
7.1 Casein Gel Formation in the presence of TGase
7.2 Reusability of the Lipase Immobilized by the TGase protocol
7.3 Stability of the Immobilized Lipase
7.4 Conclusions
8. Overall Conclusions and Recommendations
8.1 Overall Conclusions
8.2 Recommendations and Future Work
9. References
10. Appendices
10.1 Summary of Enzyme Immobilization by Chemical Methods
10.2 Summary of Literature on PEI Spacer arm in Immobilization
10.3 Purity of the lipase used in this research
10.4 Protein load of lipase immobilized cloth Via CVB protocol
10.5 Standard curve of nitrophenol concentration versus UV absorbance
10.6 UV spectra of GA Solution
10.7 UV study of preparation of PEI-polyaldehyde .
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ENZYME IMMOBILIZATION ON WOOLEN CLOTH