The ability to engineer enzymes and other proteins to any desired stability would have wide-ranging applications. Here, we demonstrate that computational design of a library with chemically diverse stabilizing mutations allows the engineering of drastically stabilized and fully functional variants of the mesostable enzyme limonene epoxide hydrolase. First, point mutations were selected if they significantly improved the predicted free energy of protein folding. Disulfide bonds were designed using sampling of backbone conformational space, which tripled the number of experimentally stabilizing disulfide bridges. Next, orthogonal in silico screening steps were used to remove chemically unreasonable mutations and mutations that are predicted to increase protein flexibility. The resulting library of 64 variants was experimentally screened, which revealed 21 (pairs of) stabilizing mutations located both in relatively rigid and in flexible areas of the enzyme. Finally, combining 10–12 of these confirmed mutations resulted in multi-site mutants with an increase in apparent melting temperature from 50 to 85°C, enhanced catalytic activity, preserved regioselectivity and a >250-fold longer half-life. The developed Framework for Rapid Enzyme Stabilization by Computational libraries (FRESCO) requires far less screening than conventional directed evolution.

We explored the use of a computational design framework for the stabilization of the haloalkane dehalogenase LinB. Energy calculations, disulfide bond design, molecular dynamics simulations, and rational inspection of mutant structures predicted many stabilizing mutations. Screening of these in small mutant libraries led to the discovery of seventeen point mutations and one disulfide bond that enhanced thermostability. Mutations located in or contacting flexible regions of the protein had a larger stabilizing effect than mutations outside such regions. The combined introduction of twelve stabilizing mutations resulted in a LinB mutant with a 23 °C increase in apparent melting temperature (Tm,app , 72.5 °C) and an over 200-fold longer half-life at 60 °C. The most stable LinB variants also displayed increased compatibility with co-solvents, thus allowing substrate conversion and kinetic resolution at much higher concentrations than with the wild-type enzyme.

The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea hrasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a chitinase from cucumber and 95% with the N-terminal sequence of the lysozyme/chitinase of Purthenocissus quinquefolia. The half-cystine residues in hevein and cucumber chitinase are located at identical positions. Hevamine is a basic protein from the lutoids (vacuoles) of rubber latex and may have a role in plugging the latex vessels and cessation of latex flow. The differences in cellular location, charge properties and sequence between hevainine and cucumber chitinase are similar to those between class I and class 11 chitinases from tobacco and other plant species. [5, 61 with affinity for chitin [7] or N-acetylglucosamine [S].Other chitinases (class 11) are acidic proteins. They belong to the pathogenesis-related proteins and are excreted by cells under several stress conditions. The sequences of acid chitinases are similar to those of the basic ones, but they lack the N-terminal hevein domain [8, Fresh latex obtained by tapping the rubber tree Hevea brasiliensis can be separated by centrifugation into three main fractions [lS]. These are a white upper layer of rubber particles, an aqueous layer containing the cytoplasma from the cells of the latex vessels and a 'bottom fraction' which consists of lutoids, vacuoles with a low internal pH that may be the equivalent of animal lysosomes [16]. Archer [I71 isolated two major basic proteins with very similar amino acid compositions from rubber latex lutoids and called them hevamine A and B, respectively. Tata et al. [18], using a similar isolation procedure, isolated the same proteins and demonstrated that both are bifunctional lysozymes/chitinases. The N-terminal sequences of hevamine, the lysozyme/chitinase of Parthenorissus and cucumber chitinase [lo, 141 are homologous or very similar.The crystallization of hevamine has been described recently [19]. In this paper we describe the complete primary structure of hevamine obtained as a bifunctional lysozyme/ chitinase from the latex of H . brasiliensis. MATERIALS A N D METHODSHevamine was isolated from freeze-dried bottom Faction of rubber latex (Hevea brasiliensis) as described by Archer [17] with several modifications [19]. Tryptic pep tidesBatches (40 mg) of hevamine were reduced and Scarboxymethylated [20J, citraconylated [21 J and digested with trypsin [22]. Digests were fractionated by gel filtration on

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␣-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an ␣-amino group. As such, they can synthesize ␤-lactam antibiotics from acyl compounds and ␤-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-Å resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an ␣/␤-hydrolase fold domain, a helical cap domain, and a jellyroll ␤-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a ␤-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an ␣-amino group on the substrate, and explains the low specificity toward the ␤-lactam nucleus.␤-Lactam antibiotics form a large family of widely applied antibacterials. Most of them are derived from a handful of naturally occurring antibiotics like penicillin G, penicillin V, and cephalosporin C by replacing their acyl groups with synthetic ones. Initially, this was achieved by chemical means but at present, enzymatic methods are preferred (1). A well known enzyme used for these conversions is penicillin acylase (EC 3.5.1.11) from Escherichia coli. This enzyme is used both for the production of the ␤-lactam nucleus 6-aminopenicillanic acid (6-APA) 1 by cleaving off phenylacetic acid from penicillin G and for the coupling of new acyl groups to 6-APA or other ␤-lactam nuclei. Penicillin acylase is, however strongly inhibited by its product phenylacetic acid (2), which must therefore be removed before coupling of a new acyl group to the ␤-lactam nucleus can take place. In addition, ␤-lactam nuclei are not very stable at the alkaline pH optimum of penicillin acylase.By contrast, ␣-amino acid ester hydrolases (AEHs) do not have these disadvantages. These enzymes catalyze the hydrolysis and synthesis of esters and amides of ␣-amino acids exclusively, and do not attack the amide bond of a ␤-lactam. They can be used to acylate a ␤-lactam using an ester as acyl donor, as shown in Fig. 1. Because the AEHs require an ␣-amino group on the substrate, they are not inhibited by phenylacetic acid (3). Together with their ability to accept various ␤-lactam nuclei without cleaving them, this makes them suitable for generating widely used antibiotics such as ampicillin, amoxicillin, and the cephalosporins cephadroxil and cephalexin. The slightly acidic pH optimum of AEH, which is beneficial for ␤-lactam stability, is another advantage of AEHs for biocatalytic applications, as is their stereospecificity toward the acyl donor (4).One of the first AEHs that was isolated and characterized is the enzyme from Xanthomonas citri (5-11). This enzyme was found to be a homotetramer with subunits of 72 kDa (6). Kinetic studies indicated the occurrence of an acyl-enzyme intermediate in the hydrolysis and acylation reactions of ␤-lactam antibiotics (3,7,12).This article reports the gene and 1.9-Å resolution ...