scholarly journals Structure-Guided Steric Hindrance Engineering of Bacillus Badies Phenylalanine Dehydrogenase for Efficient L-Homophenylalanine Synthesis

Author(s):  
Tao Wu ◽  
Xiaoqing Mu ◽  
Yuyan Xue ◽  
Yan Xu ◽  
Yao Nie

Abstract Background: Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block L-homophenylalanine (L-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading.Results: In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badies to create an enhanced biocatalyst for efficient L-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic activity. Seven superior mutants were acquired, and the optimal triple-site mutant V309G/L306V/V144G showed a 12.9-fold higher kcat/Km value than wild-type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to L-HPA in 210 min, and the specific space-time conversion was 30.9 mmoL·g−1·L−1·h−1. The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones.Conclusions: Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of L-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Tao Wu ◽  
Xiaoqing Mu ◽  
Yuyan Xue ◽  
Yan Xu ◽  
Yao Nie

Abstract Background Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. Results In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher kcat value, and accordingly a 12.9-fold higher kcat/Km value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space–time conversion was 30.9 mmol g−1 L−1 h−1. The substrate loading and specific space–time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Conclusions Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.


2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Langxing Liao ◽  
Yonghui Zhang ◽  
Yali Wang ◽  
Yousi Fu ◽  
Aihui Zhang ◽  
...  

Abstract 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 fusion 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 yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 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 fusion enzyme for the efficient biosynthesis of l-tle.


2020 ◽  
Author(s):  
Langxing Liao ◽  
Yonghui Zhang ◽  
Yali Wang ◽  
Yousi Fu ◽  
Aihui Zhang ◽  
...  

Abstract 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 fusion 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 °C, 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 fusion enzyme for the efficient biosynthesis of L-tle.


2009 ◽  
Vol 24 (18n19) ◽  
pp. 3243-3255 ◽  
Author(s):  
GERARD 't HOOFT

Matter interacting classically with gravity in 3+1 dimensions usually gives rise to a continuum of degrees of freedom, so that, in any attempt to quantize the theory, ultraviolet divergences are nearly inevitable. Here, we investigate a theory that only displays a finite number of degrees of freedom in compact sections of space-time. In finite domains, one has only exact, analytic solutions. This is achieved by limiting ourselves to straight pieces of string, surrounded by locally flat sections of space-time. Next, we suggest replacing in the string holonomy group, the Lorentz group by a discrete subgroup, which turns space-time into a 4-dimensional crystal with defects.


2005 ◽  
Vol 20 (26) ◽  
pp. 6039-6049 ◽  
Author(s):  
XIN ZHANG

A toy model based upon the q-deformation description for studying the radiation spectrum of black hole is proposed. The starting point is to make an attempt to consider the space–time noncommutativity in the vicinity of black hole horizon. We use a trick that all the space–time noncommutative effects are ascribed to the modification of the behavior of the radiation field of black hole and a kind of q-deformed degrees of freedom are postulated to mimic the radiation particles that live on the noncommutative space–time, meanwhile the background metric is preserved as usual. We calculate the radiation spectrum of Schwarzschild black hole in this framework. The new distribution deviates from the standard thermal spectrum evidently. The result indicates that some correlation effect will be introduced to the system if the noncommutativity is taken into account. In addition, an infrared cutoff of the spectrum is the prediction of the model.


1999 ◽  
Vol 77 (6) ◽  
pp. 427-446
Author(s):  
S B Phillips

A model of a spinning string with an internal coordinate index is proposed and studied. When the action for this model is taken to be diagonal in this internal coordinate space and quantized in the light-cone gauge it is found to be Lorentz covariant in four-dimensional space-time provided that the internal coordinate space is four dimensional.This combination of space-time dimension, D, and internal coordinate space dimension, N, is just one of four possible sets, the other three corresponding to D = 3, 6, and 10, precisely the same values for which it is possible to formulate Yang-Mills theories with simple supersymmetry. By comparing the number of propagating degrees of freedom at the zero-mass level in the open string bosonic and fermionic sectors it is found that a supersymmetric interpretation of this model is possible provided that all physical states in the bosonic sector have even G-parity and the ground-state spin or in the fermionic sector have positive chirality. A possible interpretation of the connection betweenthe N components of each of the D space-time coordinates is presentedon the basis that the space-time coordinates are scalars in the internal coordinate space. This interpretation would appear to be reasonable given the fact that the field variables in the Lagrangian density do not necessarily have to represent physically measurable quantities but can, instead, only represent physically measurable quantities when combined in some manner, the simplest of which being a linear combination. The Lagrangian density simply produces the equations of motion and the constraint equations for the independent variables, only linear combinations of which represent the four dimensions of physical space-time.PACS Nos.: 11.17.+y, 11.10.Qr, 1.30.Cp, 11.30.Pb


1997 ◽  
Vol 12 (32) ◽  
pp. 2421-2424 ◽  
Author(s):  
E. I. Guendelman ◽  
A. B. Kaganovich

We develop a gravitational theory where the measure of integration in the action principle is not necessarily [Formula: see text] but it is determined dynamically through additional degrees of freedom. This theory is based on the demand that such measure respects the principle of "non-gravitating vacuum energy" which states that the Lagrangian density L can be changed to L + const. without affecting the dynamics. Formulating the theory in the first-order formalism we get as a consequence of the variational principle a constraint that enforces the vanishing of the cosmological constant. The most realistic model that implements these ideas is realized in a six or higher dimensional space–time. The compactification of extra dimensions into a sphere gives the possibility of generating scalar masses and potentials, gauge fields and fermionic masses. It turns out that the remaining four-dimensional space–time must have effective zero cosmological constant.


2018 ◽  
Vol 175 ◽  
pp. 11007 ◽  
Author(s):  
Christof Gattringer ◽  
Daniel Göschl ◽  
Carlotta Marchis

We discuss recent developments for exact reformulations of lattice field theories in terms of worldlines and worldsheets. In particular we focus on a strategy which is applicable also to non-abelian theories: traces and matrix/vector products are written as explicit sums over color indices and a dual variable is introduced for each individual term. These dual variables correspond to fluxes in both, space-time and color for matter fields (Abelian color fluxes), or to fluxes in color space around space-time plaquettes for gauge fields (Abelian color cycles). Subsequently all original degrees of freedom, i.e., matter fields and gauge links, can be integrated out. Integrating over complex phases of matter fields gives rise to constraints that enforce conservation of matter flux on all sites. Integrating out phases of gauge fields enforces vanishing combined flux of matter-and gauge degrees of freedom. The constraints give rise to a system of worldlines and worldsheets. Integrating over the factors that are not phases (e.g., radial degrees of freedom or contributions from the Haar measure) generates additional weight factors that together with the constraints implement the full symmetry of the conventional formulation, now in the language of worldlines and worldsheets. We discuss the Abelian color flux and Abelian color cycle strategies for three examples: the SU(2) principal chiral model with chemical potential coupled to two of the Noether charges, SU(2) lattice gauge theory coupled to staggered fermions, as well as full lattice QCD with staggered fermions. For the principal chiral model we present some simulation results that illustrate properties of the worldline dynamics at finite chemical potentials.


RSC Advances ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 2325-2331 ◽  
Author(s):  
Yitong Chen ◽  
Baodi Ma ◽  
Songshuang Cao ◽  
Xiaomei Wu ◽  
Yi Xu

A simple and efficient process for the synthesis of optically active (S)-N-boc-3-hydroxy piperidine was developed using the “designer cells” co-expressing alcohol dehydrogenase and glucose dehydrogenase.


1998 ◽  
Vol 13 (23) ◽  
pp. 1875-1879 ◽  
Author(s):  
RICHARD J. EPP ◽  
R. B. MANN

If one encodes the gravitational degrees of freedom in an orthonormal frame field, there is a very natural first-order action one can write down (which in four dimensions is known as the Goldberg action). In this letter we will show that this action contains a boundary action for certain microscopic degrees of freedom living at the horizon of a black hole, and argue that these degrees of freedom hold great promise for explaining the microstates responsible for black hole entropy, in any number of space–time dimensions. This approach faces many interesting challenges, both technical and conceptual.


Sign in / Sign up

Export Citation Format

Share Document