A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/β-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.
The lipase-catalyzed synthesis of furan-comprising polyester oligomer diols (α,ω-telechelic diols) is reported. Oligofuranoate diols with excellent end-group fidelity and a yield of 95% were synthesized using a solvent-free two-stage polycondensation of dimethyl furan-2,5-dicarboxylate (DMFDCA) and 1,4-cyclohexanedimethanol (1,4-CHDM) using immobilized Candida antarctica Lipase B (CalB). Recycling of immobilized CalB to further decrease the production cost is successfully demonstrated. However, it showed limitation in the product yield that decreases ±20% with each additional reuse. The synthetic procedure has been scaled up, easily opening the possibility to use the developed diols in industrial polycondensations utilizing the excellent flame retardancy property and high thermal stability typical for furan-based polymers.
The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.
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