The whole-cell immobilization of D-hydantoinase-engineered Escherichia coli for D-CpHPG biosynthesis

Background: D-Hydroxyphenylglycine is considered to be an important chiral molecular building-block of antibiotic reagents such as pesticides, and β-lactam antibiotics. The process of its production is catalyzed by D-hydantoinase and D-carbamoylase in a two-step enzyme reaction. How to enhance the catalytic potential of the two enzymes is valuable for industrial application. In this investigation, an Escherichia coli strain genetically engineered with D-hydantoinase was immobilized by calcium alginate with certain adjuncts to evaluate the optimal condition for the biosynthesis of D-carbamoyl-p-hydroxyphenylglycine (D-CpHPG), the compound further be converted to D-hydroxyphenylglycine (D-HPG) by carbamoylase. Results: The optimal medium to produce D-CpHPG by whole-cell immobilization was a modified Luria-Bertani (LB) added with 3.0% (W/V) alginate, 1.5% (W/V) diatomite, 0.05% (W/V) CaCl2 and 1.00 mM MnCl2.The optimized diameter of immobilized beads for the whole-cell biosynthesis here was 2.60 mm. The maximized production rates of D-CpHPG were up to 76%, and the immobilized beads could be reused for 12 batches. Conclusions: This investigation not only provides an effective procedure for biological production of D-CpHPG, but gives an insight into the whole-cell immobilization technology.

Branch-point stoichiometry can generate weak links in metabolism: the case of glycine biosynthesis.

Although the metabolic network permits conversion between almost any pair of metabolites,this versatility fails at certain sites because of chemical constraints (kinetic,thermodynamic and stoichiometric) that seriously restrict particular conversions. We call these sites weak links in metabolism,as they can interfere harmfully with management of matter and energy if the network as a whole does not include adequate safeguards. A critical weak link is created in glycine biosynthesis by the stoichiometry of the reaction catalyzed by glycine hydroxymethyltransferase (EC 2.1.2.1), which converts serine into glycine plus one C1 unit: this produces an absolute dependence of the glycine production flux on the utilization of C1 units for other metabolic pathways that do not work coordinately with glycine use. It may not be possible,therefore,to ensure that glycine is always synthesized in sufficient quantities to meet optimal metabolic requirements.

Evidence for neuronal release of isotopically labelled glycine from the rat ventral medullary surface in vivo

Spontaneous and stimulus-induced release of isotopically labelled glycine was studied in the superfused rat dorsal or ventral medullary surface in vivo. Superfusion of the ventral medullary surface of anesthetized (urethane, 1.2 g/kg, ip) male adult Wistar rats (250-350 g) with high K+ (40 mM) surrogate cerebrospinal fluid (CSF) produced an average increase of 45 per cent over the spontaneous efflux of exogenously applied glycine (N = 5, P<0.01). In experiments in which the calcium of the CSF was replaced by an equimolar amount of magnesium, the increase in glycine efflux in response to high K+ was reduced to 15 per cent, a value not statistically different from that observed in control experiments (N = 6). Veratridine stimulation evoked a large (80 per cent) increase in glycine efflux (N = 5, P<0.001), which was inhibited by tetrodotoxin. High potassium or veratridine failed to modify spontaneous release of glycine on the dorsal medullary surface. Results obtained in control experiments showed that neither high K+ nor veratridine is effective in modifying spontaneous efflux of [(3)H]-leucine or [(3)H]-inulin on the ventral or dorsal medullary surface. These data support the hypothesis that glycine is a neurotransmitter on the ventral medullary surface and that it may be part of neural pathways involved in cardiorespiratory regulation present in this region.

Inhibición de la glicosilación no enzimática de la hemoglobina en la diabetes mellitus / Inhibition of non-enzymatic glycosilation of hemoglobin in diabetes mellitus

Se estudió el efecto inhibidor del aminoácido glicina sobre la glicosilación no enzimática de la hemoglobina en la diabetes experimental de ratas Wistar con estreptozotocina. La hemoglobina glicosilada de las ratas diabéticas fue de 4.2 ñ 0.38 por ciento y la de las diabéticas que tomaron glicina al 1 por ciento en el agua de bebida ad libitum fue de 2.90 ñ 0.37 por ciento (p = 0.00005). Un grupo de 30 personas diabéticas tipo II y 8 de tipo I tomaron glicina disuelta en agua: 20 gramos diarios (4 tomas de 5 g cada 6 horas) durante tiempos variables: de 3 hasta 56 meses. La hemoglobina glicosilada promedio de los diabéticos antes de tomar la clicina fue de 12.8 ñ 3.3 por ciento y después fue de 8.3 ñ 2.2 por ciento con un valor de p= 7 x 10-12 (prueba de rangos señalados de Wilcoxon)