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This review describes the remarkable transition in the manufacture of β‐lactam antibiotics, which is driven by the desire to reduce or eliminate the production of waste and the dependence on organic solvents. To this effect, traditional chemical procedures are gradually being replaced by biotransformations. The β‐lactam antibiotics industry has led the way in the introduction of biocatalysis in the fine chemicals industry by replacing the chemical multi‐step process for the penicillin nucleus 6‐aminopenicillanic acid (6‐APA) by an enzymatic one in the early 1990's. Recently, bioprocesses have been developed for the synthesis of the cephalosporin nucleus, 7‐aminodeacetoxycephalosporanic acid (7‐ADCA) from a penicillin precursor and will shortly be commercialized. Thirty years of research have now resulted in viable enzymatic procedures for coupling the β‐lactam nuclei with D‐phenylglycine side‐chains. The necessary adaptations in the synthesis of the side‐chain donors have likewise resulted in more efficient procedures. 1 Introduction 2 Semi‐Synthetic β‐Lactam Antibiotics: Industrial Production Prior to 1985 3 Biocatalytic Synthesis of β‐Lactam Nuclei 3.1 6‐Aminopenicillanic Acid 3.2 7‐Aminodeacetoxycephalosporanic Acid 4 Biocatalytic Routes to Side‐Chains 4.1 Synthesis of the Side‐Chain Building Blocks 4.2 Synthesis of Activated Side‐Chain Donors 5 Enzymatic Coupling of the Side‐Chains to the β‐Lactam Nuclei 5.1 Chemical Procedures 5.2 Enzymatic Coupling 5.3 Practical Procedures for Enzymatic Coupling6 Conclusion and Future Outlook

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Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier.

Candida antarctica Lipase B (CALB) formed a seemingly homogeneous solution in water, 1-ethyl-3-methylimidazolium dicyanamide ([C 2 mim][N(CN) 2 ]) or dimethyl sulfoxide (DMSO). However, dynamic light scattering (DLS) and small angle neutron scattering (SANS) demonstrated that the enzyme formed aggregates in the non-aqueous solvents. In aqueous solution, SANS measurements revealed that CALB formed cylindrical nano-structures with a diameter of 5 nm and a length of 4 nm, equivalent to the dimensions of a single CALB molecule. The enzyme also formed cylindrical structures in DMSO but the diameter was 4 nm and the length was 12 nm, indicating that the enzyme had aggregated to form dimers or trimers. In [C 2 mim][N(CN) 2 ], discshaped aggregates were formed, with an average diameter of 49 nm and a length of 3.8 nm, equivalent to the volume of 150 CALB molecules. In all cases, the hydrodynamic diameters measured by DLS matched the long axial lengths of the aggregates determined by SANS, indicating a good consistency between the two techniques. DLS measurements showed that CALB aggregates in. In all cases, the aggregation observed in the solvents was associated with loss of enzymatic activity.