Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme

Bacillus cereus e Geobacillus stearothermophilus em leite ultra alta temperatura (UAT) / Bacillus cereus and Geobacillus steartothermophilusin ultra high temperature (UHT) milk

O objetivo deste trabalho foi avaliar a presença de B. cereus e G. stearothermophilus em 100 amostras de leite UAT (integral e desnatado). O isolamento dos esporos e das células vegetativas seguiu metodologias oficiais, com pequenas modificações. B. cereus foi isolada de 7% amostras de leite UHT, de 6 diferentes marcas. As contagens máximas de células vegetativas e esporos de B. cer eus foram de 3,54 Log UFC/mL e 3,93 Log esporos/mL, respectivamente. A presença dos genes codificadores de enterotoxina não hemolítica (NHE) foi observada em 33% dos isolados e da hemolisina (HBL ) em 100% dos isolados. O gene hblA foi encontrado em 91,6 % dos isolados, porém nenhum isolado apresentou os 3 genes do complexo HBL. G. stearothermophilus foi identificada em 22,8% (34/149) dos isolados de esporo altamente resistente ao calor (HRRS), representando 18% das amostras de leite UAT e as contagens de esporos variaram de < 1Log a 3,40 Log esporos/mL.

L-Phenylalanine and L-Tyrosine Catabolism by Thermophilic Geobacillus stearothermophilus.

Aims: This study investigated the potential of soil thermophile Geobacillus stearothermophilus for the biotransformation of phenylalanine and tyrosine. Study Design: G. Stearothermopilus grows well at 65ºC and has a good potential for transformation and biodegradation of many compounds including steroids, bile acids, tryptophan and other compounds. In this study G. stearothermophilus was harvested at mid-log phase at 65ºC, on tryptone yeast extract (TYE) medium. Cells were collected by centrifugation under aseptic conditions, washed with sterile water and suspended in phosphate buffer with phenylalanine or tyrosine as sole source of carbon at 65ºC. Metabolic parameters were optimized for optimal growth of the organism utilizing aromatic amino acids as an exclusive source of carbon. Methodology: The amino acid metabolites were exhaustively extracted with methanol from freeze dried broth. The concentrated pooled extracts were analyzed by thin layer chromatography (TLC) using polar solvent systems and purification of the extracts was achieved on preparative tlc plates and GC separations. The molecular structures of purified metabolites were established through spectral data. Results: Sixteen metabolites of phenylalainine and seventeen metabolites of tyrosine were identified in this study. Tyrosine metabolism extensively produced melanin pigments that caused hitches in the purification of tyrosine metabolites. Tyr metabolites were analyzed in cells cultured for short time. Conclusion: Our data suggest that G. stearothermophilus has a good potential to metabolize aromatic amino acids yielding hydroxylated, deaminated, decarboxylated and many other products. Oxidative metabolism of phenylalanine and tyrosine by a thermophilic G. stearothermophilus is being reported for the first time.