Lijun Yang,Tiantian Zhou,Zhiyi Zhang,Wei Qi,Mengfan Wang
The enzymatic oxidation of amino acids using amino acid oxidases (AAOs) as biocatalysts for α-keto acid synthesis has garnered increasing attention due to its environmental friendliness and exceptional reaction efficiency. However, the by-product H 2 O 2 inevitably causes the decarboxylation of α-keto acids and subsequently deactivates the enzyme. Herein, we used a simple in-situ biomineralization method to immobilize Pt NPs with catalase-like activity and L-amino acid oxidase (LAAO) in to a hydrogen-bonded organic framework ( TW HOF) under a mild condition, and the synthesized LAAO-Pt@ TW HOF cascade catalytic system was applied to synthesis of α-keto acid. Compared with free LAAO, the LAAO-Pt@ TW HOF exhibited a 3.3-fold activity, well reusability and complete conversion (100 %) of L-tryptophan to indole-3-pyruvic acid was achieved. This work offers a potential application of HOF materials in constructing high-efficiency enzyme-metal coupled catalyst for cascade reactions.
l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12–24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.
Chemoenzymatic catalysis combining the traits of chemical and enzymatic catalysis provides tremendous possibilities for the design of biosynthetic pathways utilizing inorganic catalysts and enzymes. However, the efficiency of chemoenzymatic catalysis is usually governed by the synergy and compatibility of the two catalysts. Here, we report for the first time the catalase-like activity of cobalt phosphate nanocrystals (CoPs). By a one-pot biomimetic mineralization with CoPs and l-amino acid oxidase (LAAO) under a mild condition, we have fabricated a hybrid nanobiocatalyst, LAAO@CoPs, for the chemoenzymatic synthesis of α-keto acid. The as-fabricated nanobiocatalyst with directly contacted catalytic sites of the enzyme and nanozyme maximizes the substrate channeling effects for in situ chemical decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of l-tryptophan (l-Trp), thus minimizing the H2O2 accumulation and byproduct generation. Benefiting from the superiority of LAAO@CoPs, complete conversion (100.0%) of l-Trp to indole pyruvic acid is achieved, over two times higher than the yield of the free LAAO system (47.6%). Meanwhile, LAAO@CoPs show high stabilities against heat and proteolytic treatments. This work offers a new design approach for constructing a high-performance nanobiocatalyst for cascade reactions, especially for those systems with toxic or reactive intermediates.
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