L-Leucine 4-nitroanilide

A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.

This study aims to explore the potential aminopeptidases of Lysinibacillus sphaericus based on the unique metabolic characteristics of this species which cannot metabolize carbohydrates and may have a strong ability to metabolize amino acids. Fifteen peptidase-encoding genes predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli BL21, and of these genes, only Amp0279 shows a high ability to hydrolyze L-leucine-4-nitroanilide (Leu-pNA). Phylogenetic analysis, 3D-structure modeling, and enzyme assays indicated that Amp0279 should be a novel Co2+-dependent aminopeptidase belonging to the M29 family. The optimal conditions of Amp0279 were determined to be 50 °C and pH 8.0 with the addition of 100 μM Co2+, and under this condition, the specific activity of Amp0279 matched that of Flavourzyme® (3.54 × 104 vs. 3.37 × 104 U/mg for the protein ingredient of Flavourzyme®). Amp0279 is mainly expressed in the middle sporulation phase in wild-type L. sphaericus or in Bacillus subtilis under the control of the sporulation-dependent strong promoter pcry8E, which is carried by the recombinant vector pHT315-8E21b. Furthermore, the secretory expression systems based on B. subtilis and Corynebacterium glutamicum were used to enhance the soluble expression of Amp0279. Obvious expression and enzymatic activity were detected from the crude supernatant media of both host bacteria without further concentration and purification. Moreover, expression can occur in the vegetative phase in B. subtilis under the control of the Pgrac promoter. KEY POINTS: · A novel Co2+-dependent leucyl aminopeptidase Amp0279 originating from L. sphaericus was characterized. · The activity of Amp0279 as a leucyl aminopeptidase matches that of Flavourzyme® under optimal conditions. · B. subtilis- and C. glutamicum-based expression systems are built to promote secretory (soluble) Amp0279 expression.

The hydrolysis of L-leucine-p-nitroanilide by porcine kidney leucine aminopeptidase in aqueous mixed-solvent systems containing methanol, ethanol, dimethyl sulfoxide, and dimethylformamide has been investigated in the -30 to -23 degrees C temperature range. At 23 degrees C and pH* values in the 8-10 range, the enzyme is stable for over 25 h in solutions containing 50% v/v of any of these four cosolvents. Measurements of the tryptophan fluorescence of the enzyme at pH* 9.0 confirm that the enzyme is not denatured under these conditions. KM increases exponentially and kcat decreases linearly with increasing cosolvent concentration. Methanol, in particular, has a very small effect on KM. Ultrafiltration experiments demonstrate that there is no dissociation of monomers of the enzyme brought about by the presence of 50% v/v methanol or dimethyl sulfoxide. Preliminary tests with the partition method provide no evidence for an acyl-enzyme intermediate. The effect of pH* on kcat and KM in 50% v/v methanol is very similar to the effect of pH on these kinetic constants in aqueous solution. Lowering the temperature from 23 to 0 degree C does not alter the shape of the pH* profile obtained in 50% v/v methanol. The Arrhenius plot obtained in 50% v/v methanol is linear over the -30 to -23 degrees C temperature range, and the calculated energy of activation, 8.2 +/- 0.8 kcal/mol, is in good agreement with the value of 7.4 +/- 0.7 kcal/mol found for the reaction in aqueous solution. Collectively, these data indicate that methanol is the best cosolvent for cryoenzymological studies, that ethanol and dimethyl sulfoxide are also suitable cosolvents, and that the presence of any of these cosolvents at either ambient or subzero temperatures does not perturb the catalytic pathway.