Growth performance, body lipid, brood amount, and rearing environment response to supplemental neutral phytase in zebrafish (Danio rerio) diet.

The present study was to evaluate the effects of neutral phytase supplementation on growth performance, survival ratio (SR), body lipid, brood amount, and rearing environment in zebrafish. The control diet was not supplemented phytase, and three levels of phytase (500, 1000, or 1500 U kg(-1)) was added to the three other diets (named as PP500, PP1000, and PP1500). Triplicate groups (twelve 100-L tanks) of zebrafish (initial mean weight, 0.284±0.012 g) were fed twice daily (08:00 and 16:00 h) to satiation for 12 weeks. The results showed that supplemental phytase in the diet improved weight gain (60.49%, 86.63%, 99.06%, and 111.88% in control, PP500, PP1000, and PP1500) and the specific growth ratio of zebrafish (p<0.05). Dietary phytase addition increased the whole body lipid content of zebrafish. The brood amounts (116, 123, and 124 eggs in PP500, PP1000, and PP1500) of fish fed with phytase-supplemented diets were little higher than the control (mean egg was 112). The ammonia-nitrogen concentration in water of fish fed with phytase-supplemented diet was significantly lower than the control. The nitrite concentration in water was also decreased in water of fish fed with phytase-supplemented diet. The SR was increased with the increasing of dietary phytase despite no significant difference was observed among each group. The present study first suggested that neutral phytase could be applied in the zebrafish diet. Furthermore, phytase addition increased the growth, body lipid, brood amount, and SR of zebrafish, and meanwhile decreased the ammonia-nitrogen and nitrite concentrations in rearing water.

CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity.

Multicopper oxidases (MCOs) are a family of enzymes that use copper ions as cofactors to oxidize various substrates. Previous research has demonstrated that several MCOs such as MnxG, MofA and MoxA can act as putative Mn(II) oxidases. Meanwhile, the endospore coat protein CotA from Bacillus species has been confirmed as a typical MCO. To study the relationship between CotA and the Mn(II) oxidation, the cotA gene from a highly active Mn(II)-oxidizing strain Bacillus pumilus WH4 was cloned and overexpressed in Escherichia coli strain M15. The purified CotA contained approximately four copper atoms per molecule and showed spectroscopic properties typical of blue copper oxidases. Importantly, apart from the laccase activities, the CotA also displayed substantial Mn(II)-oxidase activities both in liquid culture system and native polyacrylamide gel electrophoresis. The optimum Mn(II) oxidase activity was obtained at 53°C in HEPES buffer (pH 8.0) supplemented with 0.8 mM CuCl2. Besides, the addition of o-phenanthroline and EDTA both led to a complete suppression of Mn(II)-oxidizing activity. The specific activity of purified CotA towards Mn(II) was 0.27 U/mg. The Km, Vmax and kcat values towards Mn(II) were 14.85±1.17 mM, 3.01×10(-6)±0.21 M·min(-1) and 0.32±0.02 s(-1), respectively. Moreover, the Mn(II)-oxidizing activity of the recombinant E. coli strain M15-pQE-cotA was significantly increased when cultured both in Mn-containing K liquid medium and on agar plates. After 7-day liquid cultivation, M15-pQE-cotA resulted in 18.2% removal of Mn(II) from the medium. Furthermore, the biogenic Mn oxides were clearly observed on the cell surfaces of M15-pQE-cotA by scanning electron microscopy. To our knowledge, this is the first report that provides the direct observation of Mn(II) oxidation with the heterologously expressed protein CotA, Therefore, this novel finding not only establishes the foundation for in-depth study of Mn(II) oxidation mechanisms, but also offers a potential biocatalyst for Mn(II) removal.