No abstract

An effective approach to greatly enhancing the selective secretion and expression of recombinant cytoplasmic enzymes in Escherichia coli was successfully developed through the synergistic effect of ethylenediaminetetraacetate (EDTA) and lysozyme. The method was applied to two endoglucanases (EGs) and an amylase. The optimal culture conditions of temperature and concentration of isopropyl-β-D: -1-thiogalactopyranoside (IPTG) were 23-30 °C and 0.2 mM, respectively, under which the three enzymes could be expressed in active form. Among all the chemicals tested, EDTA was found to be most suitable for enhancing the secretion of EG-I-1A into the medium. Addition of lysozyme alone had little influence on the secretion and expression. In contrast, on the basis of the addition of 5 g EDTA/L at the induction time of 12 h, the simultaneous addition of 0.15 g lysozyme/L further significantly increased the secretion and expression of the three enzymes, demonstrating the synergistic effect of EDTA and lysozyme. The production of EG-I-1A in the culture medium by adding 5 g EDTA/L and 0.15 g lysozyme/L under the optimal culture conditions of 23 °C and 0.2 mM IPTG was over 260-fold higher than that without EDTA and lysozyme under the standard conditions of 37 °C and 1 mM IPTG. In summary, the advantage of this novel cultivation approach for secretion was that not only did it selectively enhance the secretion of the proteins of interest, but also greatly increased the expression of the three enzymes by over 80 %.

A relatively high b-glucanase-producing strain Bacillus sp. III-3 was isolated from soda lakes in Neimenggu, China. This alkaliphilic strain was found to produce two thermostable b-glucanases including a novel neutral b-glucanase III-3-A and an alkaline b-glucanase III-3-B. The b-glucanases were purified to homogeneity from the culture supernatant with a two-step column chromatographic procedure. III-3-A and III-3-B had molecular mass of approximately 45 and 85 kDa, respectively. Mass spectrometry analysis indicated that III-3-A was probably different from the b-glucanases reported, whereas III-3-B showed high homology with those of family A5 alkaline b-glucanases from alkaliphilic bacilli. The optimum pH of III-3-A was about 7.0, while that of III-3-B was 8.0-10.0. Both enzymes exhibited maximum activity at 45°C and were stable up to 50°C. Ca 2+ ion stimulated the activity of III-3-A but enhanced the thermostability of III-3-B. The two enzymes were resistant to most metal ions and reagents examined.

The synthesis of optically active (R)-2-trimethylsilyl-2-hydroxyl-ethylcyanide by asymmetric trans-cyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system was achieved using (R)-oxynitrilase from loquat seed meal. Diisopropyl ether was the most suitable organic phase among the organic solvents examined. The optimal concentration of acetyltrimethylsilane, concentration of crude enzyme, volume ratio of the aqueous to the organic phase, temperature and the buffer pH value were 14 mM: , 61.4 U ml-1, 13% (v/v), 30 degrees C and 4, respectively. The substrate conversion and the product enantiomeric excess were 95% and 98% under the optimized conditions. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon counterpart.