A novel method for expression and purification of recombinant peroxidase in Spodoptera frugiperda larvae

New Biotechnology · Volume 25S · September 2009

tein secretion in C. glutamicum. In addition, the amount of pro-TG accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum surpassed that which accumulated via the Sec pathway. We conclude that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum. doi:10.1016/j.nbt.2009.06.191

2.6.088 A novel method for expression and purification of recombinant peroxidase in Spodoptera frugiperda larvae A.M. Targovnik ∗ , L.V. Romero, F.J. Wolman, O. Cascone, M.V. Miranda University of Buenos Aires, Buenos Aires, Argentina

Horseradish peroxidase (HRP) plays an important role in many biotechnological fields, including diagnostics, biocatalysis and biosensors. The aim of this work was to optimize a process for expression and purification of HRP isozyme C (HRP C) in Spodoptera frugiperda larvae. The recombinant baculovirus polyhedrin-minus was assembled by using BaculoGoldBrigth that is capable of expressing GFP and the vector pAcGP67 that encodes a sequence for the glycoprotein 67 leader peptide at the 5 end which targets the recombinant protein. This construct allows monitoring the infection visually under UV illumination. Synthetic HRPC gene was generously provided by Dr. P.E. Ortiz de Montellano of the University of California. Early fifth-instar S. frugiperda larvae were infected with the recombinant virus. A direct correlation between GFP and HRPC expression profile exists, this facilitating the selection of larvae to process from day 3-post infection. The efficiency of intrahaemocoelical infection via was 100% in all experiments. When an arbitrary threshold of 300 U/ml was set up, 50% of the larvae exceeded this reference value at day 5, 85% at day 6 and 93% at day 7. The optimum viral titre was 1.4 × 107 ufp/ml. Larval extracts at day 7 post-infection expressed 319 ± 56 mg HRPC/kg larvae while at day 6 post-infection the expression was only 137 ± 17 mg HRPC/kg larvae. The recombinant HRPC is catalytically active and its molecular mass is comparable to that of the plant enzyme (44 kDa) as judged by SDSPAGE and Western blot; on the basis of this result it can be assumed that the glycosylation degree should be similar to that of the native HRP (21.8%). HRPC was purified by ion exchange chromatography directly from the larvae extract at pH 7.0 with a yield of 80% and a purity of 70%. The process herein described is cost-effective and suitable for scaling up. doi:10.1016/j.nbt.2009.06.192

ABSTRACTS

2.6.089 Production of xanthan gum using different organic acids in culture medium M. Barboza 1,∗ , J. Leite 1 , E. Pozzi 2 , L. Pelizer 3 , M. Zaiat 2 1

Federal University of Sao Carlos, Sao Carlos, Brazil Universidade de São Paulo, Brazil 3 Universidade de Franca, Brazil 2

For over a decade, researchers have attempted to obtain chemicals, particularly acetic, propionic, butyric and hexanoic acids, through microbial processes. Those works used pure cultures primarily to produce a specific acid. In all those works, the objective was only the production of acids in high concentrations. The potential feasibility of an anaerobic bioreactor using a low content of organic matter to generate organic acids and hydrogen had been studied. The organic acids obtained can be used for several applications showing the advantage of the reuse of industrial effluents. Xanthan gum is a polymer used in several industries as pharmaceutical, food and petrochemical. Generally, it is produced by fermentation process using glucose and citric acid as substrate. But many works were done using industrial wastes as substrate in order to reduce production costs. The aim of this work was the study of the feasibility of using organic acids produced from waste treatment to obtain xanthan gum. Then polysaccharide production by Xanthomonas campestris was studied replacing the citric acid of the cultivation medium by volatile acids. Xanthan gum was produced by X. campestris (ATCC 33913). Preliminary studies were done with commercial salts, in batch process carried out in shaker at 28 ◦ C and 250 rpm. It was used three different salts replacing citric acid: (a) 0.0328 M sodium acetate, (b) sodium propionate 0.0219 M and (c) 0.0164 M sodium butyrate. The maximum productivity obtained was 0.13, 0.34 and 0.29 g xanthan gum L−1 h−1 , respectively. The best results concerning the time of the cultivation were obtained using sodium propionate and sodium butyrate. For these salts the maximum production was in 47 h while for sodium acetate it was 69 h. These results contributed for the development of this process in bioreactor in large scale using acids from waste treatment. doi:10.1016/j.nbt.2009.06.193

2.6.090 Influence of agitation, aeration and time of spore germination on the citric acid production from hemicellulosic hydrolysate in submerged fermentation R. Santos ∗ , A. Prata Universidade de São Paulo, Escola de Engenharia de Lorena, LORENA, Brazil

Citric acid is one of the most important organic acids produced by fermentation process around the world. The demand for this chemical increases every year, leading researches to develop new routes and/or substrate sources for its production. The use of hemicellulosic hydrolysates as carbon sources (mainly xylose) for

www.elsevier.com/locate/nbt S225