ABSTRACT
Glutamate dehydrogenase (GDH)a key enzyme in the nitrogen metabolism pathway catalyzes the con-version between α-ketoglutarate and glutamate reversibly using NAD(P)H as a cofactor. Based on genomic stud-ies,it was concluded that SHJG_7666 was a potential GDH in Streptomyces hygroscopicus 5008(S5008),and its expression level in vivo was positively correlated with the biosynthesis of an important aminocyclol compound vali-damycin. Phylogenetic tree analysis showed that the S5008 SHJG_7666 GDH belonged to the Glu/Leu/Phe/Val dehydrogenase family,with conserved glutamate-α-ketoglutarate binding domain and the classical GXGXXG dinu-cleotide binding motif. Further homologous modeling and structural comparison revealed that SHJG_7666 con-tained conserved Lys60,Lys78and Asp120catalytic functional sites and ligand binding residues Ser36,Gly38,Gln119 and Asp166,Asn300,Ala330. Moreover,recombinant expression of SHJG_7666 in E. coli and in vitro enzyme activity demonstrated that glutamate dehydrogenase can convert ammonium salt to glutamate with pH and temperature being optimal at 7. 5 and 37 °C respectively. Enzyme activity under optimum reaction condition has Kmvalue of (25. 3 ±9. 1)μmol/L and kcatof (3 ±0. 8)×10 -5s-1for the substrate α-ketoglutarate. Results of this study further improved the catalytic activity of SHJG_7666,thus laying the foundation for the ultimate increase of vali-damycin production.
ABSTRACT
Objective:The aim is to establish L-glutamate specific aminotransferase-L-glutamate dehydrogenase coupling 96-well high throughput screening method,which is applied to molecular evolution of aminotransferase WecE from E.coli.Methods:An optical assay for aminotransferase catalytic activity based on aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time.Mutants library of WecE is obtained by sitedirected saturation mutagenesis.Positive mutants can be screened out through 96-well preliminary screening and flask second screening.Results:The target transamination reaction is coupled with L-glutamate dehydrogenase indicative reaction system which consists of 0.5 U/ml enzyme loading and 0.4 mmol/L NADH.A positive mutant Y321F whose catalytic activity increases 3.4 fold compared to that of wild type is screened out in Tyr 321 saturation mutagenesis library of WecE.Conclusion:An accurate high throughput screening method with weak background interference is established.It offers feasible solution for molecular evolution of L-glutamate specific aminotransferase.
ABSTRACT
Objective To investigate the clinical curative effect of the surgical method of transferring the radialis proper digital nerve of damaged index finger and its dorsal branch to repair the thumb nerve evulsion.Methods 13 patients with thumb nerve evulsion were treated.There were 8 males and 5 females with an average age of 28 years (ranged 18-52 years old).The injuries were caused by machine twist (8 cases),gear(4 cases),electric saw (1 case).And thumb rotational avulsion amputation (10 cases),thumb incompleteness amputation(3 cases).The time from injury to admission was 1-3.5 h (mean 2.2 h).The average time from injury to admission was (1.1 ± 1.5) h.The amputate level of skin was at the juxtra-articular of metacarpophalangeal joint.The amputate level of bone was at the base of proximal phalanx (6 patients) and metacarpophalangeal joint (4 patients),interphalangeal joint(3 patients).Using transferring the radialis proper digital nerve of index finger without reimplantation and its dorsal branch to.repair the both side injuries of thumb nerve evulsion.According to routine method to repair digital bone,veins,arteries and tendons.Results All 13 chases were followed up for 6 months to 2 years and 7 months postoperatively,with an average of 22 months.The wounds and incisions at donor sites were healed by first intention.All 13 cases of thumb reimplantation were successful.Two-point discrimination of ulnaris finger pulp was 2 to 6mm,average 4.2mm,and the radialis was 5 to 9mm,average 7.8mm.Sensory function was rated as S4(the ulnaris in 12 cases and the radialis in 2 cases) and S3 + (the radialis in 11 cases and the ulnaris in 1 cases).Conclusion Transferring the radialis proper digital nerve of damaged index finger and its dorsal branch to the digital nerve on the neighboring thumb is a simple and effective method to restore sensory function of the pulp.