Peter Remler

Biocatalytic Friedel–Crafts Alkylation Using Non‐natural Cofactors

The formation of C C bonds is a central aspect of synthetic organic chemistry. However, in biocatalysis only few enzymes capable to perform this reaction are known, among which aldolases, transketolases, and hydroxynitril lyases have been investigated thoroughly. Some have even found their way into industrial applications. Friedel–Crafts alkylation is a classic organic reaction of great importance. However, in particular for large scale application, this transformation is ecologically very critical and regiospecific monoalkylation is difficult to achieve. Therefore, an environmentally friendly and selective alternative would be highly desirable. In nature methyl groups are selectively introduced into reactive aromatic rings by methyltransferases (Mtases), in particular with S-adenosyl-lmethionine (SAM) as the cofactor. Furthermore, enzyme-catalyzed reactions are important for access to isoprenoids. Also, prenylation of aromatic rings has been performed. For phenylalanine ammonia lyases a Friedel–Crafts-type mechanism has been proposed. Recently, it has been shown that besides the methyl group other alkyl, alkenyl, and alkinyl groups can be introduced into S-adenosyl-l-homocystein. These modified cofactors of transferases were used for a sequence-specific alkylation of DNA. Having cofactor and modified cofactors in hand, we investigated the possibility of alkylation of aromatic substrates, thus transferring the biosynthesis into the laboratory (Scheme 1). Aminocoumarins are antibiotics produced by some Streptomyces species and are targets for the methyl transfer from the natural cofactor SAM. The Mtase A and B are involved in the biosynthesis of the antibiotics coumermycin A1 [7] (produced by Streptomyces rishiriensis) and novobiocin (produced by Streptomyces spheroides ; Scheme 1). SAM analogues were synthesized by modifying the strategy published by Klimašauskas, Weinhold, and co-workers. S-Adenosyl-l-homocysteine (SAH) was alkylated by seven different alkyl bromides using formic acid as the solvent and AgOTf as a Lewis acid activator and catalyst. We observed quantitative conversion in less than 2 days (average reaction time 24 h; Table 1). The chemical synthesis of SAM analogues results in approximately 1:1 diastereomeric mixtures at the sulfonium center. In the first screenings the diastereomers were separated by preparative HPLC and used as cofactors for the alkylation of coumarin compound 3a. Both epimers were accepted by the enzymes NovO and CouO Scheme 1. C-Mtases involved in the biosynthesis of the antibiotics coumermycin A1 in Streptomyces rishiriensis and Novobiocin in Streptomyces spheroides.

Hydrolysis of polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s−1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s−1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene.

C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli.

Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40°C and pH 7-11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40°C and pH=9.5 resulted in K(M)=0.23 ± 0.01 mM and k(cat)=167.5 ± 3.6s(-1). MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.

Extracellular serine proteases from Stenotrophomonas maltophilia: screening, isolation and heterologous expression in E.coli.

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45°C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17±2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents.

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an α-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.