Cortical visual function in the rd12 mouse model of Leber Congenital Amarousis (LCA) after gene replacement therapy to restore retinal function.

One eye of rd12 mice received a sub-retinal injection of a vector carrying normal human RPE65 cDNA at post-natal day 18, and at 6- and 13-months of age. Electroretinograms (ERGs) and visual-evoked potentials (VEPs) were recorded to luminance, and to spatially and temporally modulated stimuli to assess the consequences of delayed treatment on visual pathway function. Early treatment resulted in better overall retinal rescue and better rescue of cone-mediated function. VEPs to low temporal frequency luminance modulation were well preserved at all but the oldest treatment age and corresponded to predictions based on the amount of retinal rescue. In contrast, VEPs to high frequency spatially and temporally modulated stimuli were impaired even at the earliest age. These results provide further support that early treatment in human LCA will have the most hope for optimal visual performance.

Production and partial characterization of thermostable and calcium-independent alpha-amylase of an extreme thermophile Bacillus thermooleovorans NP54.

AIM: An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS: The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS: Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY: The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles.

The extensive efforts to screen thermophilic fungi and bacteria, isolated from various environmental samples, have resulted in the selection of Thermomucor indicae-seudaticae, Geobacillus thermoleovorans NP33 and G. thermoleovorans NP54 for the production of glucoamylase, amylopullulanase and alpha-amylase, respectively. Submerged and solid-state fermentation processes were optimized for maximizing the secretion of glucoamylase by T. indicae-seudaticae. The production of amylopullulanase and alpha-amylase by NP33 and NP54 in submerged fermentation was also optimized. Glucoamylase was optimally active at pH 7.0 and 60 degrees C and was shown to saccharify soluble as well as raw starches. Amylopullulanase and alpha-amylase exhibited optima at pH 7.0 and 100 degrees C and saccharified starch efficiently. Differential inhibition and action on mixed substrates clearly suggested that there are two separate active sites for alpha-amylase and pullulanase activities of amylopullulanase. Both alpha-amylase and amylopullulanase are high maltose-forming and Ca(2+)-independent. These amylolytic enzymes have been shown to be useful in starch saccharification alone and in combination.