RESUMO
Xylitol-phosphate dehydrogenase (XPDH) genes from several Gram-positive bacteria were isolated and expressed in Bacillus subtilis. The substrate specificities of the recombinant XPDH enzymes were compared and it was found that the XPDH enzymes of Lactobacillus rhamnosus and Clostridium difficile had the highest selectivity towards D-xylulose 5-phosphate. Expression of these two XPDH enzymes in D-ribulose and D-xylulose producing B. subtilis strain resulted in strains of B. subtilis capable of converting D-glucose into xylitol at around 23% yield.
Assuntos
Bacillus subtilis/metabolismo , Proteínas Recombinantes/metabolismo , Desidrogenase do Álcool de Açúcar/genética , Xilitol/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Fermentação , Engenharia Genética , Glucose/metabolismo , Lacticaseibacillus rhamnosus/enzimologia , Lacticaseibacillus rhamnosus/genética , Ribulosefosfatos/metabolismo , Xilose/metabolismoRESUMO
D-arabinitol 1-phosphate (Ara-ol1-P), a substrate for D-arabinitol-phosphate dehydrogenase (APDH), was chemically synthesized from D-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD(+) and NADP(+) were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.
Assuntos
Pentosefosfatos/síntese química , Pentosefosfatos/metabolismo , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo , Bacillus/enzimologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Enterococcus/enzimologia , Enterococcus/genética , Enterococcus/metabolismo , Cinética , NAD/metabolismo , NADP/metabolismo , Oxirredução , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Xilulose/metabolismoRESUMO
A novel method for D-arabitol production with a metabolically engineered Bacillus subtilis strain is described. A known transketolase-deficient and D-ribose-producing mutant of B. subtilis (ATCC 31094) was further modified by disruption of its rpi (D-ribose phosphate isomerase) gene to create a D-ribulose- and D-xylulose-producing B. subtilis strain. Expression of the D-arabitol phosphate dehydrogenase gene of Enterococcus avium in the D-ribulose- and D-xylulose-producing strain resulted in a strain of B. subtilis capable of converting D-glucose to D-arabitol with a high yield (38%) and little by-product formation.
Assuntos
Bacillus subtilis/metabolismo , Enterococcus/enzimologia , Melhoramento Genético/métodos , Glucose/metabolismo , Engenharia de Proteínas/métodos , Desidrogenase do Álcool de Açúcar/metabolismo , Álcoois Açúcares/metabolismo , Bacillus subtilis/genética , Enterococcus/genética , Desidrogenase do Álcool de Açúcar/genética , Álcoois Açúcares/isolamento & purificaçãoRESUMO
An enzyme with a specificity that has not been described previously, D-arabitol-phosphate dehydrogenase (APDH), has been purified from cell lysate of Enterococcus avium. SDS/PAGE indicated that the enzyme had a molecular mass of 41+/-2 kDa, whereas a molecular mass of 160+/-5 kDa was observed under non-denaturing conditions, implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have a narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into xylulose 5-phosphate and ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD(+) and NADP(+) were accepted as cofactors. Based on the partial protein sequences, the APDH gene was cloned. Homology comparisons place APDH within the medium-range dehydrogenase family. Unlike most members of this family, APDH requires Mn(2+) but no Zn(2+) for enzymic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system. Both biochemical evidence and protein sequence homology comparisons indicate that similar enzymes are widespread among the Gram-positive bacteria. Their apparent biological role is to participate in arabitol catabolism via the 'arabitol phosphate route', similar to the ribitol and xylitol catabolic routes described previously.