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Abstracts / Journal of Biotec

VI4-P-013

Combinational biosynthesis of a fluorescent alpha-subunit phy-cocyanin (spirulina platensis) in Escherichia coli

Guan Xiangyu 1,2,∗, Qin Song 1,2

1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao, PRChina2 Ocean University of China, Qingdao, PR China

E-mail address: xiangyu guan@yahoo.com.cn (G. Xiangyu).

Among numerous oxidizing species, the reactive oxygen ones play amajor part in diverse important pathological processes (Finkel andHolbrook, 2000). Phycocyanin (PC) is a biliprotein of blue–greenalgae, and a variety of nutritional and pharmacological values of PChave been reported (Romay et al., 1998).

A vector harboring two cassettes was constructed: cpcA encod-ing apo-alpha-PC of Spirulina platensis (Sp) along with cpcE-cpcFof Synechocystis sp. PCC6803 (S6) encoding lyases required for theattachment of bilin chromophores to apo-alpha-PC in one cassette;ho1-pcyA of S6 encoding enzymes required for the conversion ofheme to phycocyanobilin (PCB) in another cassette (Tooley et al.,2001; Guan et al., 2007). After induction with IPTG, the trans-formants with final expression vector pCDF-cpcA(Sp)-cpcE-cpcF,ho1-pcyA exhibited a blue color. A fluorescent holo-alpha-PC of Spwith His-tag (rHHPC) was biosynthesized in Escherichia coli BL21by only one expression vector. The constant feeding mode wasadopted, and transformant reached the biomass of rHHPC up to0.55 g/L broth in 5-L bench scale. The recombinant rHHPC waspurified by Ni2+ affinity column conveniently. The purified rHHPCshowed correct molecular weight on SDS-PAGE gel and emittedorange fluorescence by UV excitation. The maximum peaks ofrHHPC in absorbance and fluorescence emission spectrum were at621 nm and 651 nm, which was similar to those of native C-PC. Fur-thermore, inhibition effect on hydroxyl and peroxyl radicals makesrHHPC as a potent antioxidant, whose function may be partiallyresponsible to the anti-tumor effect. The IC50 values of rHHPC were277.526 �g/ml against hydroxyl radicals and 20.833 �g/ml againstperoxyl radicals.

This study provides an efficient method for large-scale produc-tion of the fluorescent phycobiliproteins. The rHHPC with severalunique qualitative and quantitative features exhibits promisingapplications in therapeutic and fluorescent tagging fields insteadof native PC in practice (Sun et al., 2003).

References

Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing.Nature 408, 239–247.

Guan, X.Y., Qin, S., Su, Zh.L., Zhao, F.Q., Ge, B.S., Li, F.C., Tang, X.X., 2007. Combinationalbiosynthesis of a fluorescent cyanobacterial holo-�-phycocyanin in Escherichiacoli by using one expression vector. Appl. Biochem. Biotech. 142, 52–59.

Romay, C., Armesto, J., Remirez, D., Gonzalez, R., Ledon, N., Garcia, I., 1998. Antioxi-dant and anti-inflammatory properties of C-phycocyanin from blue-green algae.Inflamm. Res. 47, 36–41.

Sun, L., Wang, S.M., Chen, L.X., Gong, X.Q., 2003. Promising fluorescent probes fromPhycobiliproteins. IEEE J. Quantum. Elect. 9, 177–188.

Tooley, A.J., Cai, Y.A., Glazer, A.N., 2001. Biosynthesis of a fluorescent cyanobacterialC-phycocyanin holo-alpha subunit in a heterologous host. Proc. Natl. Acad. Sci.U.S.A. 98, 60–65.

doi:10.1016/j.jbiotec.2008.07.1325

gy 136S (2008) S558–S576 S563

VI4-P-014

Algicidal activity of Achromobacter sp. (strain YZ) isolated fromyellow sea: An assessment with bloom causing cyanobacteriumMicrocystis aeruginosa

Hui Wang ∗, Zhaopu Liu ∗, Surya Kant Mehta, Gengmao Zhao

Key Laboratory of Marine Biology of Jiangsu Province, Nanjing Agri-culture University, Nanjing 210095, China

E-mail address: sea@njau.edu.cn (H. Wang).

With objective to control algal blooms an algae-lytic bacterium(Zhao et al., 2005), temporarily named as YZ, was isolated from theyellow sea and was test for killing of bloom causing cyanobacteriumMicrocystis aeruginosa. Based on biochemical and biophysical char-acteristics (IMCAS, 1978) and 16SrRNA gene sequencing the isolatewas identified as Achromobacter sp. The isolate YZ was foundvery effective in lysing and decomposing laboratory-grown Micro-cystis. The results showed that initial bacterial and algal cellsdensities strongly influence the removal rates of chlorophyll-a.The greater the initial bacterial cells density the faster was thedegradation of chlorophyll-a. However algal cells density had recip-rocal effect on degradation of chlorophyll-a by the bacterium. Wefurther found that lipid peroxidation, measured in term of malon-dialdehyde content, significantly increased from 0.14098 �mol L−1

to 0.252 �mol L−1 (78.74%) when alga was co-cultivated with YZ.It suggested that isolate YZ caused lysis of Micorcystis via induc-tion of lipid peroxidation. The growth of alga was strongly inhibitedby the bacterial filtrate previously autoclaved and treated by pro-tein K indicating that the algae-lytic substance produced by strainYZ was extracellularly produced, non proteinaceous and thermo-stable (indirect attack) (Imai et al., 2001). The environmental factorssuch as temperature, illumination, pH have influence on the killingeffects of the bacterium: the best lytic effects were achieved atlower temperatures and in the dark. The ability of YZ to lysis Micor-cystis decreased in the following order of pH: 3 > 9 > 5 > 7.

References

Imai, I., Sunahara, T., Nishikawa, T., 2001. Fluctuations of the red tide flagellatesChatonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. Inthe Seto Inland Sea, Japan. Mar. Biol. 138, 1043–1049.

Institute of Microbiology of Chinese Academy of Sciences, 1978. The General Methodof Bacterial Identification. Science Press, Bejing.

Zhao, P.C., Pu, Y.P., Yin, L.H., 2005. Development of research on algicidal bacteria andits evaluation. J. Southeast Univ. 24 (3), 202–206.

doi:10.1016/j.jbiotec.2008.07.1326

VI4-P-015

Carbonylcyanide m-chlorophenylhrazone (CCCP) regulatedhydrogen production by marine green algae Platymonassubcordiformis subjected to light-dark-light treatment

Yunbin Fu 1,2,∗, Zhaoan Chen 1, Zhen Guo 1,2, Hongbin Lu 1, XingjuYu 1, Wei Zhang 1,2

1 Marine Bioproducts Engineering Group, Dalian Institute of ChemicalPhysics, Chinese Academy of Sciences, Dalian 116023, China2 Graduate School of the Chinese Academy of Sciences, Beijing 100049,China

E-mail address: weizhang@dicp.ac.cn (W. Zhang).

Platymonas subcordiformis was found to produce hydrogen via pho-tolysis of seawater (Guan et al., 2004). In order to improve hydrogenproduction, the effect of light-dark-light treatment on the durationand yield of hydrogen evolution was investigated in the process reg-