Bookedited by Fraser Armstrong.
Contents:
Front Cover; Enzymes of Energy Technology; Copyright; Contents; Contributors; Preface; Chapter One: Purification of fully active and crystallizable photosystem II from thermophilic cyanobacteria;
1. Introduction;
2. Growth and harvest of cells;
3. Isolation of thylakoid membranes;
4. Isolation of crude PSII particles;
5. Purification of PSII dimers;
6. Characterization of the purified PSII core dimers;
7. Crystallization of purified PSII dimers; Acknowledgments; References; Chapter Two: Production and manipulation of blue copper oxidases for technological applications;
1. Introduction
2. Heterologous production of fungal laccases2
.1. Cloning; 2.1
.1. Site-directed mutagenesis; 2.1
.2. Yeast recombination; 2
.2. Transformations; 2.2
.1. S. cerevisiae; 2.2
.2. A. niger; 2
.3. Screening for laccase secreting transformants in S. cerevisiae and A. niger; 2.3
.1. Screening of clones from deep-well or microtiter plate cultures; 2.3
.2. Screening of laccase producing transformants on petri dish plates; 2.3
.3. Screening of activity on 96-well plate; 2.3
.4. ELISA screening; 2
.4. Fermentation in A. niger; 2.4
.1. Preparation of spore inoculum and storage 2.4
.2. Production in bioreactor and growth parameter
s3. General purification procedure and characterization; 3
.1. Purification procedure; 3.1
.1. Assay procedures; 3.1
.2. Laccase purification procedure;
4. Applications; 4
.1. General procedure for an oriented covalent grafting of the laccase surface; 4.1
.1. Preparation of laccase variants for single-site anchoring; 4.1
.2. Reductive amination of lysine; 4.1
.3. Evaluation of the grafting; 4
.2. Hybridization with materials;
5. Conclusions; Acknowledgment; References; Further reading Chapter Three: Production and spectroscopic characterization of lytic polysaccharide monooxygenase
s1. Introduction;
2. Expression and purification of LPMOs; 2
.1. Recombinant expression and purification from the E. coli periplasm; 2
.2. Recombinant expression and purification from the E. coli cytoplasm; 2
.3. Recombinant expression and purification from yeast; 2
.4. Recombinant expression and purification from fungi;
3. EPR spectroscopy; 3
.1. EPR sample preparation for LPMOs; 3
.2. Multifrequency collection; 3
.3. Simulation procedures; Acknowledgments; References; Further reading Chapter Four: A novel overproduction system for the structural determination of a proton-pumping hydrogen-producing [NiFe ..
.1. Introduction; 1
.1. Hydrogen catalysis in nature; 1
.2. Escherichia coli hydrogenase-2 genetic and structural arrangement; 1
.3. First approaches to overexpression of a [NiFe]-hydrogenase;
2. Overproduction of fully formed holo-Hyd-2; 2
.1. Molecular biology strategies for the overproduction of Hyd-2; 2.1
.1. Design of overexpression plasmid pO; 2.1.1
.1. Equipment; 2.1.1
.2. Reagents and buffers; 2.1.1
.3. Procedures; 2.1
.2. Design of overexpression strain HJ001-hyp