The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-subtilisin. Of them, the structure and function of Tk-subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was approximately 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-subtilisin and bacterial subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.

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Copper-doped zinc oxide nanoparticles were successfully synthesized by grinding copper acetate and zinc acetate powder with different starting molar ratios in combined with sodium hydroxide. The effect of initial copper and zinc molar ratios on the product samples was investigated and discussed. Relevant ligand coordination type of reactant acetate salt precursors and product samples were investigated by Fourier transform infrared spectroscopy (FTIR). The particle shapes and surface morphologies were characterized by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Phase structures of prepared samples were studied by x-ray powder diffraction (XRD) and x-ray absorption near-edge spectroscopy (XANES) was applied to investigate the local structure of Cu and Zn environment atoms. The results demonstrate that the, particle size of as-synthesized products affected by copper concentration in the precursor trend to gradually decreases from nanorod shape with diameter around 50–100 nm to irregular particle structure around 5 nm associated with an increase in the concentration of copper in precursor. Moreover, it is noticed that shape and morphology of the products are strongly dependent on Cu:Zn ratios during the synthesis. Nanocrystallines Cu-doped ZnO by the substitution in Zn site with a high crystallization degree of hexagonal wurtzite structure were obtained. This synthesis technique is suggested as a potential effective technique for preparing copper zinc oxide nanoparticles with various atomic ratio in wide range of applications.

Kojic acid is a secondary metabolite produced by some strains of Aspergillus spp and has been exploited commercially in food and cosmetic products. The objective of this work was to optimize the cultivation conditions of Aspergillus oryzae ATCC 10124 prior to triggering the fermentation process using organic broken rice noodles (OBRN) as a carbon source. Trials with various carbon and nitrogen sources show that 10% glucose and 0.05% yeast extract with ammonium sulfate was favorable for A. oryzae to produce kojic acid (1.58 g/L at day 4 of cultivation). Initial pH and agitation rate significantly affected the kojic acid formation. The maximum quantities of kojic acid were obtained when the pH of the medium was at 2.5, and shaking was at 200 rpm, at 1.60 g/L and 1.65 g/L, respectively. When the carbon source was changed to OBRN, the substrate was saccharified by alpha-amylase and glucoamylase, and 100% of the hydrolysate was used to replace glucose for the kojic acid fermentation. Kojic acid content increased when using OBRN, at 1.52 g/L, and was comparable to that yielded from the fermentation with glucose (1.58 g/L).

Pro‐Tk‐SP from Thermococcus kodakaraensis consists of the four domains: N‐propeptide, subtilisin (EC 3.4.21.62) domain, β‐jelly roll domain and C‐propeptide. To analyze the maturation process of this protein, the Pro‐Tk‐SP derivative with the mutation of the active‐site serine residue to Cys (Pro‐Tk‐S359C), Pro‐Tk‐S359C derivatives lacking the N‐propeptide (ProC‐Tk‐S359C) and both propeptides (Tk‐S359C), and a His‐tagged form of the isolated C‐propeptide (ProC*) were constructed. Pro‐Tk‐S359C was purified mostly in an autoprocessed form in which the N‐propeptide is autoprocessed but the isolated N‐propeptide (ProN) forms a stable complex with ProC‐Tk‐S359C, indicating that the N‐propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC‐Tk‐S359C, instead of the ProN:ProC‐Tk‐S359C complex. The C‐propeptide was autoprocessed and degraded when ProC‐Tk‐S359C was incubated at 80 °C in the absence of Ca2+. However, it was not autoprocessed in the presence of Ca2+. Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca2+ suggests that the C‐propeptide becomes highly resistant to proteolytic degradation in the presence of Ca2+. We propose that Pro‐Tk‐SP derivative lacking N‐propeptide (Val114‐Gly640) represents a mature form of Pro‐Tk‐SP in a natural environment. The enzymatic activity of ProC‐Tk‐S359C was higher than (but comparable to) that of Tk‐S359C, suggesting that the C‐propeptide is not important for activity. However, the Tm value of ProC‐Tk‐S359C determined by far‐UV CD spectroscopy was higher than that of Tk‐S359C by 25.9 °C in the absence of Ca2+ and 7.5 °C in the presence of Ca2+, indicating that the C‐propeptide contributes to the stabilization of ProC‐Tk‐S359C.