Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.
l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and β-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU μg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.
The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.