Jonathan Woodward

Fungal and other β-d-glucosidases — Their properties and applications

Abstract β- d -Glucosidase (β- d -glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β- d -glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β- d -glucosidase, which has high stability and can be recovered and reused. The current state of β- d -glucosidase biotechnology is described.

Biotechnology: Enzymatic production of biohydrogen

Although in theory the amount of hydrogen that could be generated from renewable sources of energy such as cellulose (a polymer of glucose) is vast, only 16–24% of the maximum stoichiometric yield of hydrogen from glucose (about 12 mol H2 per mol glucose) is typically achieved by biological methods. Here we show that the enzymes of the oxidative pentose phosphate cycle can be coupled to hydrogenase purified from the bacterium Pyrococcus furiosus, one of only a few hydrogenases that use NADP+ as the electron carrier, to generate 11.6 mol H2 per mol glucose-6-phosphate. Hydrogen produced by this pathway is the major product, unlike that produced by intermediate metabolic pathways of bacterial fermentation, and therefore has important practical implications for biohydrogen production.

Palladium-bacterial cellulose membranes for fuel cells

Bacterial cellulose is a versatile renewable biomaterial that can be used as a hydrophilic matrix for the incorporation of metals into thin, flexible, thermally stable membranes. In contrast to plant cellulose, we found it catalyzed the deposition of metals within its structure to generate a finely divided homogeneous catalyst layer. Experimental data suggested that bacterial cellulose possessed reducing groups capable of initiating the precipitation of palladium, gold, and silver from aqueous solution. Since the bacterial cellulose contained water equivalent to at least 200 times the dry weight of the cellulose, it was dried to a thin membranous structure suitable for the construction of membrane electrode assemblies (MEAs). Results of our study with palladium-cellulose showed that it was capable of catalyzing the generation of hydrogen when incubated with sodium dithionite and generated an electrical current from hydrogen in an MEA containing native cellulose as the polyelectrolyte membrane (PEM). Advantages of using native and metallized bacterial cellulose membranes in an MEA over other PEMs such as Nafion 117 include its higher thermal stability to 130 degrees C and lower gas crossover.

Comparison of techniques for enzyme immobilization on silicon supports

Abstract Enzyme immobilization onto silicon substrates has been investigated by five different coupling procedures. The methods included covalent coupling either through a metal link reagent or silane reagents containing pendant amino or epoxide linkers, an entrapment technique using a thin layer of gelatin, or an adsorption technique using poly- l -lysine. These immobilization procedures were evaluated using glucose oxidase and a simple spectrophotometric method employing Fenton’s reagent. Retention of enzyme activity and surface loading were assessed. The immobilization techniques were also evaluated by electron microscopy to characterize the evenness of the surface coatings. All of the covalent coupling procedures led to surface loadings, approaching 1 pmol mm −2 ; however, the surfaces appeared irregular on a microscopic scale. The poly- l -lysine adsorption technique provided the smoothest surface. With the exception of the entrapment technique, all immobilization procedures provided immobilized enzyme that retained >75% activity after several weeks of storage.

The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy

Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented.

Enzymatic production of biohydrogen.

Although in theory the amount of hydrogen that could be generated from renewable sources of energy such as cellulose (a polymer of glucose) is vast, only 16–24% of the maximum stoichiometric yield of hydrogen from glucose (about 12 mol H2 per mol glucose) is typically achieved by biological methods. Here we show that the enzymes of the oxidative pentose phosphate cycle can be coupled to hydrogenase purified from the bacterium Pyrococcus furiosus, one of only a few hydrogenases that use NADP+ as the electron carrier, to generate 11.6 mol H2 per mol glucose-6-phosphate. Hydrogen produced by this pathway is the major product, unlike that produced by intermediate metabolic pathways of bacterial fermentation, and therefore has important practical implications for biohydrogen production.

Synergism in cellulase systems

Abstract Synergism between different cellulase components exists when they are acting simultaneously on insoluble cellulosic substrates. However, the degree of synergism observed appears to depend on several factors, including the type of substrate and the nature of a particular cellulase component. Cross-synergism between the cellobiohydrolase and endoglucanase components from different sources also occurs. The mechanism of synergism between components is not yet understood, but there is a clear relationship between the ability of an endoglucanase component to be adsorbed on the surface of cellulose and its potential to synergistically interact with a cellobiohydrolase. Also, the degree of synergism is greatest when the substrate is not saturated with enzyme.

Methods of immobilization of microbial cells

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Flocculation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Adsorpt ion of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Immobil izat ion by cell entrapment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Immobil izat ion of microbial cells by metal-l ink/chelation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Gold coated lanthanide phosphate nanoparticles for targeted alpha generator radiotherapy.

Targeted radiotherapies maximize cytotoxicty to cancer cells. In vivo α-generator targeted radiotherapies can deliver multiple α particles to a receptor site dramatically amplifying the radiation dose delivered to the target. The major challenge with α-generator radiotherapies is that traditional chelating moieties are unable to sequester the radioactive daughters in the bioconjugate which is critical to minimize toxicity to healthy, non-target tissue. The recoil energy of the 225Ac daughters following α decay will sever any metal-ligand bond used to form the bioconjugate. This work demonstrates that an engineered multilayered nanoparticle-antibody conjugate can deliver multiple α radiations and contain the decay daughters of 225Ac while targeting biologically relevant receptors in a female BALB/c mouse model. These multi-shell nanoparticles combine the radiation resistance of lanthanide phosphate to contain 225Ac and its radioactive decay daughters, the magnetic properties of gadolinium phosphate for easy separation, and established gold chemistry for attachment of targeting moieties.

The fate of MAb-targeted Cd125mTe/ZnS nanoparticles in vivo

INTRODUCTION Nanoparticles (NP) have potential as carriers for drugs and radioisotopes. Quantitative measures of NP biodistribution in vivo are needed to determine the effectiveness of these carriers. We have used a model system of radiolabeled quantum dots to document the competition between efficient vascular targeting and interaction of the NP with the reticuloendothelial (RE) system. METHODS We have prepared (125m)Te-labeled CdTe NP that are capped with ZnS. Te-125m has a half-life and decay characteristics very similar to those for (125)I. The synthesized particles are stable in aqueous solution and are derivatized with mercaptoacetic acid and then conjugated with specific antibody. To evaluate specific targeting, we used the monoclonal antibody MAb 201B that binds to murine thrombomodulin expressed in the lumen of lung blood vessels. The MAb-targeted NP were tested for targeting performance in vivo using single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging, tissue autoradiography and standard organ biodistribution techniques. Biodistribution was also determined in mice that had been depleted of phagocytic cells by use of clodronate-loaded liposomes. RESULTS Cd(125m)Te/ZnS NP coupled with MAb 201B retained radioisotope and antibody activity and accumulated in lung (>400% injected dose [ID]/g) within 1 h of intravenous injection. Control antibody-coupled NP did not accumulate in lung (<10% ID/g) but accumulated in liver and spleen. Images from microSPECT/CT and autoradiography studies of the targeted NP document this specific uptake and demonstrate uniform distribution in lung with minor accumulation in liver and spleen. Within a few hours, a large fraction of lung-targeted NP redistributed to spleen and liver or was excreted. We hypothesized that NP attract phagocytic cells that engulfed and removed them from circulation. This was confirmed by comparing biodistribution of targeted NP in normal mice versus those depleted of phagocytic cells. In mice treated with clodronate liposomes, accumulation of NP in liver was reduced by fivefold, while accumulation in lung at 1 h was enhanced by approximately 50%. By 24 h, loss of the targeted NP from lung was inhibited by several-fold, while accumulation in liver and spleen remained constant. Thus, the treated mice had a much larger accumulation and retention of the NP at the target site and a decrease in dose to other organs except spleen. CONCLUSION Nanoparticles composed of CdTe, labeled with (125m)Te and capped with ZnS, can be targeted with MAb to sites in the lumen of lung vasculature. In clodronate-treated mice, which have a temporary depletion of phagocytic cells, accumulation in liver was reduced dramatically, whereas that in spleen was not. The targeting to lung was several-fold more efficient in clodronate-treated mice due to larger initial accumulation and better retention of the MAb-targeted NP at that site. This model system indicates that targeting of NP preparations is a competition between the effectiveness of the targeting agent and the natural tendency for RE uptake of the particles. Temporary inhibition of the RE system may enhance the usefulness of NP for drug and radioisotope delivery.