Dean Brady

Advances in enzyme immobilisation

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the β-substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted β-hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of α-hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.

Microbial nitrilases: versatile, spiral forming, industrial enzymes

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a αββα–αββα sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

Screening of commercial enzymes for the enantioselective hydrolysis of R,S-naproxen ester

This study focused on the identification of suitable lipase or esterase activity for enantiomeric resolution of ( R,S)-naproxen. For an economically viable reaction the enantiomeric ratio (E) should preferably be >100, while maximising the conversion will reduce the mass of material that requires racemisation and recycling. Hence the aim was to find an enzyme that yields ( S)-naproxen with an enantiomeric excess of more than 98%, a substrate conversion in excess of 40% of the racemate, and an E of >100. (R,S)-Naproxen ethyl ester (NEE) (50 mg) was used as substrate for enzyme hydrolysis reactions at 37 ◦ C for 4 h. Biocatalyst screening was performed in buffered aqueous solvent on a 1 ml scale. The reactions were stopped with 2 ml MeCN, filtered through cotton wool and analysed by HPLC to determine the percentage m/m and R/S ratio. Eight commercially available enzymes were selected for optimisation of enantioselectivity through statistically designed experiments where the reaction conditions were varied. ChiroCLEC-CR from Altus and ESL001-01 from Diversa provided acceptable enantiomeric excess, but only ChiroCLEC-CR met the specification set for the enantiomeric ratio ( E).

Spherezymes: A novel structured self-immobilisation enzyme technology

BackgroundEnzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.ResultsThrough addition of protein cross-linking agents to a water-in-oil emulsion of an aqueous enzyme solution, structured self-immobilised spherical enzyme particles of Pseudomonas fluorescens lipase were formed. The particles could be recovered from the emulsion, and activity in aqueous and organic solvents was successfully demonstrated. Preliminary data indicates that the lipase tended to collect at the interface.ConclusionThe immobilised particles provide a number of advantages. The individual spherical particles had a diameter of between 0.5–10 μm, but tended to form aggregates with an average particle volume distribution of 100 μm. The size could be controlled through addition of surfactant and variations in protein concentration. The particles were robust enough to be recovered by centrifugation and filtration, and to be recycled for further reactions. They present lipase enzymes with the active sites selectively orientated towards the exterior of the particle. Co-immobilisation with other enzymes, or other proteins such as albumin, was also demonstrated. Moreover, higher activity for small ester molecules could be achieved by the immobilised enzyme particles than for free enzyme, presumably because the lipase conformation required for catalysis had been locked in place during immobilisation. The immobilised enzymes also demonstrated superior activity in organic solvent compared to the original free enzyme. This type of self-immobilised enzyme particle has been named spherezymes.

Diamination by N-coupling using a commercial laccase

Nuclear diamination of p-hydrobenzoquinones with aromatic and aliphatic primary amines was catalysed by an immobilised commercial laccase, Denilite II Base, from Novozymes. The amine and the p-hydrobenzoquinone was reacted under mild conditions (at room temperature and at 35 degrees C) in a reaction vessel open to air in the presence of laccase and a co-solvent to afford, exclusively, the diaminated p-benzoquinone. These compounds may have potential antiallergic, antibiotic, anticancer, antifungal, antiviral and/or 5-lipoxygenase inhibiting activity.

One-Pot Laccase-Catalysed Synthesis of 5,6-Dihydroxylated Benzo[b]furans and Catechol Derivatives, and Their Anticancer Activity

A commercial laccase, Suberase® from Novozymes, was used to catalyse the synthesis of 5,6‐dihydroxylated benzo[b]furans and catechol derivatives. The yields were, in some cases, similar to or better than that obtained by other enzymatic, chemical or electrochemical syntheses. The synthesised derivatives were screened against renal (TK10), melanoma (UACC62), breast (MCF7) and cervical (HeLa) cancer cell lines. GI50, TGI and LC50 are reported for the first time. Anticancer screening showed that the cytostatic effects of the 5,6‐dihydroxylated benzo[b]furans were most effective against the melanoma (UACC62) cancer cell line with several compounds exhibiting potent growth inhibitory activities (GI50 = 0.77–9.76 µM), of which two compounds had better activity than the anticancer agent etoposide (GI50 = 0.89 µM). One compound exhibited potent activity (GI50 = 9.73 µM) against the renal (TK10) cancer cell line and two exhibited potent activity (GI50 = 8.79 and 9.30 µM) against the breast (MCF7) cancer cell line. These results encourage further studies of the 5,6‐dihydroxylated benzo[b]furans for their potential application in anticancer therapy.

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre‐enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).

Defining a process operating window for the synthesis of 5-methyluridine by transglycosylation of guanosine and thymine

This paper describes a high yielding coupled enzymatic reaction using Bacillus halodurans purine nucleoside phosphorylase (PNP) and E. coli uridine phosphorylase (UP) for synthesis of 5-methyluridine (5-MU) by transglycosylation. Key parameters such as reaction temperature, pH, reactant loading, reactor configuration and enzyme loading were investigated. A guanosine conversion of 95% and a 5-MU yield of 85% were achieved at 1l scale, with a productivity of 10 g l⁻¹ h⁻¹.

High-yielding cascade enzymatic synthesis of 5-methyluridine using a novel combination of nucleoside phosphorylases

Abstract A novel combination of Bacillus halodurans purine nucleoside phosphorylase (BhPNP1) and Escherichia coli uridine phosphorylase (EcUP) has been applied to a dual-enzyme, sequential, biocatalytic one-pot synthesis of 5-methyluridine from guanosine and thymine. A 5-methyluridine yield of >79% on guanosine was achieved in a reaction slurry at a 53 mM (1.5% w/w) guanosine concentration. 5-Methyluridine is an intermediate in synthetic routes to thymidine and the antiretroviral drugs zidovudine and stavudine.