One‐step Synthesis of α‐Keto Acids from Racemic Amino Acids by A Versatile Immobilized Multienzyme Cell‐free System

Abstract

The elevated value of α‐keto acids has pushed scientists to explore more efficient and less expensive alternatives for their synthesis. In this work, an immobilized tri‐enzyme system that produced α‐keto acids in “one‐pot” from l‐ or racemic mixtures of diverse amino acids was presented. The system combined a broad‐spectrum amino acid racemase (BsrV), a d‐amino acid oxidase (DAAO) and catalase (CAT). BsrV racemized l‐amino acids into their d‐enantiomers, DAAO catalyzed the stereospecific oxidative deamination of the d‐amino acids into their corresponding α‐keto acids, ammonium ion, and H2O2. Finally, CAT converted the inactivating H2O2 into H2O and O2, which can be reused by the oxidase reaction. BsrV thermal stability was improved 3,300‐fold by immobilizing the enzyme on glyoxyl‐activated agarose beads. DAAO and CAT were co‐immobilized on agarose beads functionalized with glutaraldehyde groups for enhancing their stabilities and eliminating H2O2 in a much more effective way. To show the versatility of this system, racemic mixtures of amino acids were converted in their corresponding α‐keto acids. The coupling of the three immobilized enzymes permitted conversions of approximately 99 % through a dynamic kinetic resolution process. This system conserved 100 % of its initial effectiveness after 8 reaction cycles. Collectively, our innovative tri‐enzyme system for the synthesis of α‐keto acids opens the door for a cheapening in the production of many pharmaceutical and cosmetics.

Co-immobilization multienzyme nanoreactor with co-factor regeneration for conversion of CO2

“…Generally, the stability of enzyme could be improved by multipoint or multisubunit immobilization due to preventing subunit dissociation, decreasing aggregation, enhancing enzyme rigidification and resistance to inhibitors or chemicals, and et al [6,7]. Recent years, multiple enzyme cascade reactions have a significant role for the production of many compounds at an industrial level because they permit to perform very complex reactions [8][9][10][11]. In particular, multi-enzymatic conversion of CO 2 has exhibited good application prospects.…”

Enzyme production ofd-gluconic acid and glucose oxidase: successful tales of cascade reactions

“…This may be a key point if the intermediate product/products are unstable and can be destroyed if they are not rapidly modified. [223][224][225][226][227][228][229][230][231][232] Co-immobilization may be also necessary if cofactors and enzymes are immobilized in porous supports, as the enzyme will only have access to the cofactors located in the same porous particles. [233][234][235][236] On the negative side, it can be noted that in a co-immobilized biocatalyst, all enzymes should be immobilized using the same surface, and the stability of the least stable enzyme will determine the stability of the whole co-immobilized enzyme biocatalyst.…”

Co-immobilized Alcohol Dehydrogenase and Glucose Dehydrogenase with Resin Extraction for Continuous Production of Chiral Diaryl Alcohol

“…Co-immobilization could facilitate in situ removal of by-products, and is essential for reactions that require cofactor regeneration [14,15]. Co-immobilization of the main and regeneration enzymes can also facilitate the mobility of the co-factor between two enzymes.…”