l-amino acid ligase: A promising alternative for the biosynthesis of l-dipeptides

Wang, Tao; Zhang, Yufei; Ning, Li-xiao; Wang, Yifan; Liu, Xiaohuan; Li, Rui; Chen, Xiang-e

doi:10.1016/j.enzmictec.2020.109537

Cited by 17 publications

(7 citation statements)

References 62 publications

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“…A recent review on LALs identified both the establishment of ATP regeneration and immobilization of these enzymes as important endeavors, but noted that these have not been explored in the scientific literature so far (Wang et al, 2020). The only exceptions were a small number of patents describing the combination of PPKs with LALs (Hashimoto et al, 2006;Yu et al, 2021;Zhu et al, 2019) and one patent on immobilization of an LAL (Yu et al, 2020).…”

Section: Aim Of This Thesis 20 2 Aim Of This Thesismentioning

confidence: 99%

Biocatalytic Production of Bioactive Dipeptides

Bordewick¹,

Berger²

2023

Lebensmittelchemie

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Dipeptides are a promising substance class with a large variety of bioactivities. N‐terminal ar‐ ginyl dipeptides (Arg‐X) have been shown to exhibit both salt‐taste enhancing (Arg‐Ser among others) and antihypertensive properties (Arg‐Phe). A novel biocatalytic strategy for the specific synthesis of these dipeptides is the application of L‐amino acid ligases like RizA from B. subtilis, which enables production of Arg‐X dipeptides directly from their amino acids. However, challenges for an industrial application are the dependence on the expensive cofactor ATP (adenosine triphosphate), low product yields, formation of the undesired side product Arg Arg and the potentially high enzyme costs.In order to reduce the need for ATP, a strategy for ATP regeneration from acetyl phosphate was established using acetate kinase from E. coli (AckA). Up to 5.9 g/L Arg‐Ser were produced, corresponding to an ATP efficiency of 23 g Arg‐Ser per gram ATP (without regeneration max. 0.5 g Arg‐Ser/g ATP). Next, variants of RizA were created to alter the substrate specificity. A total of 21 RizA variants were created by site‐directed mutagenesis of eight positions in the substrate binding pocket. Additional 14 variants were created by combination of different mutations. Variants with improved activity and specificity were identified for all five examined amino acid combinations (Arg + Arg, Asp, Ser, Ala, Phe). The largest change was observed for K83F_S156A, which improved the product formation of Arg‐Phe six‐fold and improved specificity more than tenfold. Finally, co immobilization of RizA and selected variants with AckA was performed to enable reusability of this biocatalytic system. Immobilisates of T81F_A158S produced up to 3.0 g/L Arg Ser and immobilisates of K83F_S156A produced up to 3.8 g/L Arg‐Phe and could both be reused several times.This thesis represents the first extensive scientific investigation into establishing ATP regeneration for an L‐amino acid ligase and co‐immobilization of such a biocatalytic system, and substantially expanded the knowledge about engineering the substrate specificity of L amino acid ligases.

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Section: Aim Of This Thesis 20 2 Aim Of This Thesismentioning

confidence: 99%

Biocatalytic Production of Bioactive Dipeptides

Bordewick¹,

Berger²

2023

Lebensmittelchemie

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“…Recently, two promising enzymes, namely, α-amino acid ester acyltransferase (AET) [5] and L-amino acid ligase (Lal) [6] , have been identified as being capable of catalyzing the enzymatic biosynthesis of Ala-Gln. SsAET was discovered in Sphingobacterium siyangensis AJ2458 [5] , which uses L-alanine methyl ester hydrochloride (AlaOMe) and unprotected Gln to create Ala-Gln directly.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, Lals are considered to be a viable tool for industrial dipeptide synthesis. With Lals, several systems have been developed for the biosynthesis of Ala-Gln [12][13][14] . In particular, to date, it has been reported that the Lal fromBacillus altitudinis (BABD) might display the highest catalytic performance in the biosynthesis of Ala-Gln [15] .…”

Section: Introductionmentioning

confidence: 99%

Cell@MOF nanocomposites with improved catalytic performance for the enzymatic biosynthesis of Ala-Gln

chen,

Di,

Liu

et al. 2024

Preprint

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Recently, zeolitic imidazolate frameworks (ZIFs) with tunable pore sizes and large surface areas have been widely used as effective supports for enzyme immobilization. A bienzyme system for the biosynthesis of L-alanyl-L-glutamine (Ala-Gln) with effective ATP regeneration was established in this study. Cells (E. coli BL21 (DE3)) expressing L-amino acid ligase (Lal) from Bacillus altitudinis (BABD) and polyphosphate kinase (PPK) were immobilized on ZIF-8 using a one-step molecular organic framework (MOF)-embedded approach. The immobilized bienzyme system, named BP@ZIF-8, was successfully fabricated and characterized using Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The resulting BP@ZIF-8 exhibited high thermostability at 50 °C and high pH stability in neutral and alkaline reaction buffers. BP@ZIF-8, for example, lost little activity after 150 minutes in carbonate buffer solution (pH 10.0). However, BP@ZIF-8 displayed poor pH stability in acidic environments. Furthermore, after seven successive uses, it still maintained 58.9% of their initial activity, exhibiting satisfactory reusability. After 14 hours, the yield of Ala-Gln catalysed by BP@ZIF-8 was around 14.56 mM, which represented an increase of roughly 14.3% above that catalysed by free cells. These findings showed that BP@ZIF-8 has a great deal of potential for practical Ala-Gln biosynthesis.

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“…l -amino acid ligases (LALs; EC 6.3.2.28) are a relatively novel enzyme class that synthesizes dipeptides from their respective amino acids. They belong to the ATP-grasp superfamily and hydrolyze ATP to ADP and P i to catalyze the amide bond formation through an acyl phosphate intermediate [ 1 ]. The first LAL ywfE (also called BacD) was discovered in B. subtilis in 2005 [ 2 ].…”

Section: Introductionmentioning

confidence: 99%

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides

Bordewick¹,

Berger²,

Ersoy³

2022

Molecules

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The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

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l-amino acid ligase: A promising alternative for the biosynthesis of l-dipeptides

Cited by 17 publications

References 62 publications

Biocatalytic Production of Bioactive Dipeptides

Biocatalytic Production of Bioactive Dipeptides

Cell@MOF nanocomposites with improved catalytic performance for the enzymatic biosynthesis of Ala-Gln

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides

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