Silicate bacteria or Bacillus mucilaginosus, which is a special Bacillus species producing a rich variety of exo-polysaccharides and have some capability of dissolving potassium, phosphorus and silicate from minerals, may have wide potential uses in agriculture and industry. In order to increase its capability on hydrolyzing phytate phosphorus so as to make a microbial fertilizer, a transgenic Bacillus strain highly secreting phytase was constructed by the particle bombardment method using a phytase secreting expression vector pSP43 with a mini-tn5 transposon and a phytase expression cassette. In this study, the vector pSP43 was triumphantly transferred into the Bacillus mucilaginosus, and three transgenic strains with a stable copy of phytase expression cassette integrated into the chromosomal of the Bacillus mucilaginosus by tn5 transposition were selected. The phytase activity of engineering strains obtained increased 36-46times in comparison with the start strain D4B1. This is the first report made a transgenic Bacillus strain by the particle bombardment method successfully.

Responses of aquicultured lupin and tomato plants to low P concentrations in the growth media were compared. Under the low P condition, the lupin and tomato plants exhibited similar responses though the rates of the responses were different. These responses included an increased secretion of organic acids, Apase and phytase by roots. However, the following differences were apparent. (1) The increase of Apase secretion was higher in lupin roots than in tomato roots while the increase of phytase secretion was reverse. (2) The increase of organic acid secretion was considerably higher in lupin than that in tomato. (3) The external threshold P concentration in growth media to induce the responses was considerably lower in lupin (approximately 10 μmol/L) than in tomato (300 μmol/L P). (4) The physiological threshold P content (PTPC), an internal P content to induce the response, was considerably higher in tomato (4.2 mg P.g-1) than in lupin (1.5 mg P.g-1), indicating that the P requirement of tomato for normal growth was quite higher than that of lupin plant. It was considered that the high P requirement of tomato for normal growth was the most important factor in the low adaptive ability to P deficient conditions. The secretion of Apase, phytase and organic acids by roots provides a set of adaptive mechanisms by which plants can enhance the availability of P and therefore alter the nutrient status in the rhizosphere.

In this study we purified both acid phosphatases (Apase) secreted from tomato and lupin roots, and compared the properties of these two enzymes. The secretory Apases from tomato and lupin showed the following similar properties. 1) Both enzymes were homodimers consisting of two identical subunits, each with a molecular weight of approximately 68 kilodaltons (kD) in tomato and 72kD in lupin. 2) The enzymes were stable in the pH range of 4-9. 3) The enzymes showed an optimum temperature of 37-40℃ for their activity and were stable at temperatures below 60℃. 4) The enzymes exhibited a omparable affinity for p-nitrophenyl phosphate (the apparent Km values were 2.7-3.0×10-5M). On the other hand, there were some slight differences in the isoelectric point, optimum pH, specific activity, substrate specificity, and inhibitory effect of metal ions between the two enzymes. herefore, it was considered that the most important difference in the root ability of the two plants to hydrolyze organic phosphorus may depend mainly on the amount of Apase secreted from a unit amount of roots.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The hytase was successfully separated from the major acid phosphatase to an eletrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (Km=38μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45℃, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.