Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis

Abstract

Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD ? as cofactor over NADP ? . The purified recombinant P. pini GDH displayed a specific activity of 247.5 U/mg. The enzyme was stable in the pH range 4-8.5 and exhibited excellent thermostability till 50 °C for 24 h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD ? -dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2 h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.

Overexpression and biochemical characterization of a carboxyspermidine dehydrogenase from Agrobacterium fabrum str. C58 and its application to carboxyspermidine production

“…Pyridine nucleotide regeneration is vital in NAD(P) + ‐dependent oxidoreductase‐catalyzed reactions, such as alcohol dehydrogenase, 34 lactate dehydrogenase 35 and 2,3‐butanediol dehydrogenase 36 . In previous studies, glucose was added as a cheap reducing agent donor to generate NAD(P)H by glucose dehydrogenase (GDH) in a cascade reaction system 37‐39 . Formate dehydrogenase (FDH) catalyzed the oxidation of formate to CO 2 coupled to the reduction of NAD(P) + to NAD(P)H. The advantages of the practically irreversibly reaction, broad pH optimum (pH 6.0–9.0) and easy removal from the main product gave FDH an important application value in the NAD(P)H regeneration system 40,41 …”

“…36 In previous studies, glucose was added as a cheap reducing agent donor to generate NAD(P)H by glucose dehydrogenase (GDH) in a cascade reaction system. [37][38][39] Formate dehydrogenase (FDH) catalyzed the oxidation of formate to CO 2 coupled to the reduction of NAD(P) + to NAD(P)H. The advantages of the practically irreversibly reaction, broad pH optimum (pH 6.0-9.0) and easy removal from the main product gave FDH an important application value in the NAD(P)H regeneration system. 40,41 Use of the engineered CS-1 strain as the whole-cell biocatalyst for C-Spd production from putrescine and L-Asa has been reported for the first time.…”

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…pET vectors were by far the most commonly employed (in 66% of cases), and they were primarily utilized in conjunction with DE3 strains since the T7 RNA polymerase gene of DE3 is required for efficient synthesis of sequences downstream of T7/Lac hybrid promoters present in many pET plasmids [ 93 ]. pET28 was the most popular choice of vector, featuring in approximately 31% of the studies that utilized pET derivatives, followed by pET21 [ 8 , 29 , 36 , 94 – 96 ], pET32 [ 97 , 98 ], pET24 [ 99 , 100 ], and pET15 [ 48 , 77 ] (Fig. 1 D).…”

“…The placement of rare codons with an mRNA region can promote stability in addition to ribosomal stalling allowing extra time for folding of problematic peptide regions [ 157 ]. These studies purchased synthetic genes from commercial manufacturers including GenScript USA Inc. [ 132 , 158 , 159 ], Invitrogen [ 96 , 160 ], Sloning BioTechnology GmbH, and Synbio Technologies [ 47 ] that carry out codon optimisation on their products as a default service. The Graphical Codon Usage Analyser [ 41 ] and the Genescript Rare Codon Analysis Tool [ 72 ] were used for in-house codon analysis [ 6 , 40 ].…”

Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli

“…In the area of NADH regeneration, glucose dehydrogenase enzyme remains the most popular and dominant. [5][6][7][8] Other NADH regeneration systems include formate dehydrogenase, [9][10][11] and NADH oxidase. [12][13][14] To be effective and useful, a cofactor regeneration system should deliver high yield of cofactor per molecule of substrate, and less often noted, be of relatively fast kinetics.…”