Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides.

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.

Proximate analysis and physico-chemical properties of flour from the seeds of the China chestnut, Sterculia monosperma Ventenat.

The aim of this study was to evaluate nutritional composition of China chestnut seeds, Sterculia monosperma Vent. and analyze the physico-chemical properties of flour from the seeds. The results obtained on proximate analysis of China chestnut seeds, S. monosperma, revealed that they contained mostly carbohydrate (73.7% dm), followed by fat (12.0% dm), protein (7.8% dm), fiber (5.5% dm) and ash (1.0% dm). They have a relatively high content of potassium (12.3 mg g(-1) dm) following by phosphorus (2.30 mg g(-1) dm), magnesium (1.87 mg g(-1) dm), sulfur (0.88 mg g(-1) dm) and calcium (0.14 mg g(-1) dm). The fatty acids profile was found to be composed of mainly palmitic (42%) and oleic acids (34%), with general long-chain fatty acids the other significant component by mass (13%). Glutamic acid (17.4%), aspartic acid (12.5%) and arginine (12.5%) were the three major amino acid constituents. The purified seed starch was investigated for its morphological, starch content and physico-chemical properties, such as amylose content, swelling power, solubility and pasting properties. The starch granules were quite round, about 10-15 micron diameter and composed of more than 35% (w/w) of amylose. The pasting properties of flour from the seeds of S. monosperma revealed that gelatinization began at 72.6-73.2 degrees C and the maximum viscosity in the holding period at 95 degrees C was 633 BU. Interestingly and potentially of use, was that the viscosity at the cooling period was more than two-fold higher than that in the holding period.