Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units/mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.     

Effects of Mg2+, NaCl, citric acid, and other factors on synthesis and accumulation of β-carotene inDunaliella salina

The effect of Mg²⁺, NaCl and citric acid on the accumulation of β-carotene inDunaliella Salina was studied. The experimental results showed that 10.5 mmol/L Mg²⁺, 5 mol/L NaCl, 3 μmol/L citric acid, and CO₂ are favorable forDunaliella Salina cell growth and β-carotene accumulation. After 144 h culture under the above conditions, theDunaliella Salina biomass increased by 7.18 times; β-carotene reached 9.61%. Project 89023990 supported by the Shandong Natural Science Fund.     

Effect of nitrate and phosphate on accumulation of β-carotene isomers inDunaliella salina

Dunaliella salina, a halotolerant unicellular green alga, can accumulate a large amount of β-carotene under certain environmental conditions. The isomers of β-carotene extracted fromD. salina cultured in medium with different nitrate and phosphate concentrations were analysed by HPLC with Alox-T alumina column. At least six isomers were found in different proportions depending on the culture media's nitrate and/or phosphate concentrations. Nitrate and/or phosphate deficiency was conducive to the accumulation of total cis isomers but not of all trans isomer. It is suggested that 1 mmol/L KNO₃ and 0.1 mmol/L KH₂ PO₄ are favourable for accumulation of total cis β-carotene. Contribution No. 2090 from the Institute of Oceanology, Chinese Academy of Sciences. This project was supported by the National Natural Science Foundation of China, Grant No. 38970587.     

Immunotoxicity effect of benzo[α]pyrene on scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 μgL⁻¹) on scallop (Chlamy farreri) immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL⁻¹ B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]P. Bacteriolytic activity in 0.1μgL⁻¹ B[α]P treatment group and antibacterial activity in 0.1 μgL⁻¹ and 0.5 μgL- B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 μgL⁻¹ B[α]P treatment group (P>0.05). Thus, B[α] has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms.     

Effects of diet and body size on phosphorus utilization of Liza haematocheila T. ＆ S.

A 21-d laboratory experiment was conducted to study, the phosphorus （P） utilization of two different diets by redlip mullet Liza haematocheila T. ＆ S. Sand-filtered water in salinity 30 and temperatare 25℃ was used. Twenty-nine fish individuals were divided into three groups： 11 to group 1 （G1） fed on diet 1, 11 to group 2 （G2） fed on diet 2, and 7 to contrast group. Diet 1 was a commercial feed, more valuable in nutrition than diet 2 that similar to natural detritus. The results show the intake phosphorus （IP） of G1 was significantly higher than that of G2, and both increased linearly with body size at a certain amount of diet. The retention phosphorus （RP） in fish of G1 was lower than G2. The relationship between retention phosphorus and body size was positive and stronger in G2. Significant difference in faecal phosphorus （FP） was found between G1 and G2. Body size significantly impacted the excretion phosphorus （EP） in G1 but G2. The loss of intake phosphorus in G1 was 10.83-20.27 mg per g fish weight gain, higher than that in G2 for 6.63-9.56. Of the phosphorus, about 10% was allocated into growth, 50% in faeces, and the rest lost in excretion. The main part of phosphorus was lost in faeces but excretion. The phosphorus budget of the fish could be described as 100IP = 7.40RP ＋ 47.39FP ＋ 36.63EP （Diet 1） or 100IP = 11.93RP ＋ 56.64FP ＋ 21.76EP （Diet 2）.     

Effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta

The effect of dietary phosphorus on the growth and body components of juvenile Synechogobius hasta was determined. Different percentages of dietary phosphorus (0.63, 0.77, 0.93, 1.06, 1.22 and 1.36) were tested by feeding the fish (body weight, 15.81 g ± 0.32 g; 20 individuals each group; 3 groups each percentage) at a surplus of 5%–10% above satiation for 35 d. Dietary phosphorus did not significantly affect the specific growth rate, feed intake, feed conversion ratio and protein efficiency rate. Nitrogen retention was found to be the highest in fish fed the diet containing 1.06% of phosphorus; however, this was not significantly different from other diets. Fish fed the diet containing 0.93% of phosphorus showed the highest phosphorus retention; similar phosphorus retention rates were found in fish fed the diets containing 0.77% and 1.06% of phosphorus. Fish fed the diet containing the highest percentage of dietary phosphorus were found to contain the least whole body lipid, lower than fish fed other diets (P<0.05). The protein content increased from 18.59% to 19.55% (although not significant) with the decrease of body lipid content (P>0.05). The contents of the whole body ash, whole body phosphorus and vertebrae phosphorus increased with dietary phosphorus percentage up to 1.06 (P<0.05), reaching a plateau after that. Dietary phosphorus did not significantly influence the muscle components (protein, lipid and moisture). Condition factor and hepatosomatic index were the highest in fish fed the diet containing 0.63% of dietary phosphorus; however, this was not significantly different from those of other diets. The second-order polynomial regression of phosphorus retention against dietary phosphorus identified a breakpoint at 0.88% of dietary phosphorus. However, the dietary requirement of phosphorus for maintaining maximum phosphorus storage determined by broken-line analysis of the contents of whole body phosphorus, and ash and vertebrae phosphorus was 1.06% of the diet.     

Effect of ration, temperature and body weight on growth of black porgy,Sparus macrocephalus (B.)

Growth of black porgy,Sparus macrocephalus, was studied experimentally at four ration levels from starvation to satiation and four temperatures ranging from 14.8 to 26.8°C. At maximum rations, the specific growth rate (SGR) increased with temperature and was a negative linear function of body weight. The relationship between SGR and ration levels was a decelerating curve. Stepwise regression was used to develop the predictive models for the specific growth rates. Maintenance rations and optimum rations both increased with increased temperature. The relationship between maintenance, optimum rations and body weight was influenced by temperature. Conversion efficiencies increased with ration from zero at maintenance ration to a peak at the optimum ration, then declined with further increased ration. Contribution No. 3127 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39370542 supported by NSFC and study also supported by National Key Laboratory in the Institute of Hydrobiology, Chinese Academy of Sciences.     

Facile Conversion of Alcohols to Olefins by Tosylation and Subsequent SiO2-promoted β-elimination

A convenient and effective procedure was developed for the conversion of alcohol to olefin by tosylation and subsequent β-elimination promoted by silica gel in this study. Treatment of the alcohols with p-toluenesulfonyl chloride in pyridine at 0℃ affords tosylates which undergo β-elimination with silica gel in dichloromethane or chloroform at room temperature, yielding olefinswith high productivity.