Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins

Hölsch, Kathrin; Weuster‐Botz, Dirk

doi:10.1042/ba20100143

Cited by 30 publications

(24 citation statements)

References 55 publications

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“…It had been reported that the fusion of two multimeric enzyme could lead to the formation of multimeric protein network [41] which may cause steric hindrance of active domain. Soluble bi-functional enzyme compose of two multimeric enzyme have reported to form oligomers [29,42]. LeuDH and FDH in our study usually formed octamer and dimer, respectively.…”

Section: The Influence Of Peptide Linker On the Thermal Stability Of supporting

confidence: 47%

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Zhang

Wang

et al. 2017

Engineering in Life Sciences

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This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.

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Section: The Influence Of Peptide Linker On the Thermal Stability Of supporting

confidence: 47%

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Zhang

Wang

et al. 2017

Engineering in Life Sciences

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“…3, 4). It had been reported that the fusion of two multimeric enzymes could lead to the formation of a multimeric protein network [30] and the bifunctional enzyme compose of two multimeric enzymes could form oligomers [31]. In this work, LeuDH and GDH usually form in octamer and tetramer respectively, then the implementation of GDH-R3-LeuDH could form a multimeric state which explained the enhance of stability of the fusion enzyme.…”

Section: Discussionmentioning

confidence: 64%

Construction and Characterization of a Novel Glucose Dehydrogenase-Leucine Dehydrogenase Bifunctional Enzyme for the Biosynthesis of L-Tert-Leucine

Liao

Zhang

Wang

et al. 2020

Preprint

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Background: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully.Results: In this work, a novel bifunctional enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yeild of L-tle by GDH-R3-LeuDH was all enhanced by 2-fold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/d in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30℃, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH bifunctional enzyme for the efficient biosynthesis of L-tle.

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“…Flexible linkers are typically incorporated in cases where the fused domains require a certain degree of movement or interaction (Kim et al, 2000;Blazyk and Lippard, 2004;Jiang et al, 2005;Wang et al, 2007;Lu and Feng, 2008). Most commonly used flexible linker sequences primarily consist of Gly and Ser residues ("GS" linker) (Huston et al, 1988;Kavoosi et al, 2007;Hölsch and Weuster-Botz, 2010). (G 4 S 1 ) n is one such widely used sequence.…”

Section: Discussionmentioning

confidence: 99%

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

Xu¹,

Ding²,

Cui³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS) 1 , L2: (GGGGS) 2 , and L3:(GGGGS) 3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins 19361Linker length affects HSA-fused ONC in P. pastoris ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 19360-19370 (2015) were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

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Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins

Cited by 30 publications

References 55 publications

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

Construction and Characterization of a Novel Glucose Dehydrogenase-Leucine Dehydrogenase Bifunctional Enzyme for the Biosynthesis of L-Tert-Leucine

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

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