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.

Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55°C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55°C and retained more than 70% of its activity after incubation at 70°C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

Eficacia da descontaminacao de residuos biologicos infectantes de laboratorios de microbiologia apos tratamento termico por autoclavacao / Efficacy of the decontamination of biological infectious waste after thermal treatment by autoclaving

A pesquisa foi realizada no Laboratório Central de Saúde Pública de Minas Gerais com o objetivo de validar o processo de descontaminação de resíduos infectantes do subgrupo A1 e identificar possíveis falhas no procedimento preliminar à sua disposição final. Foram avaliados tanto a descontaminação dos resíduos totalmente descartáveis acondicionados em sacos plásticos termorresistentes quanto o processo de descontaminação dos resíduos reutilizáveis provenientes do Laboratório de Tuberculose, acondicionados em caixas metálicas. Enquanto os resultados obtidos no primeiro estudo indicaram uma deficiência considerável no tratamento dos resíduos, no segundo caso a eficácia foi comprovada. Medidas preventivas e corretivas foram propostas e adotadas como consequência deste trabalho, e são aqui descritas.

Laboratory studies were performed at the Central Laboratory of Public Health of Minas Gerais in order to validate the process of infectious waste decontamination (subgroup A1) from the public health service and identify possible flaws in the procedure preliminary to its final disposal. We evaluated both the decontamination of disposable waste packed in thermo-resistant plastic bags as well and the decontamination process of reusable waste from the Tuberculosis Laboratory packed in metallic boxes. The results of the first study indicated a significant deficiency in waste treatment, while in the second case efficacy was demonstrated. Preventive and corrective measures were proposed and adopted as a result of this work and are described herein.

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.

16α-Hydroxycholic Acid: In Vivo Microbial Transformation Product of Cholic Acid.

Aims: This study describes the transformation of cholic acid to hydroxylated cholic acid metabolites that could not be easily synthesized. Study Design: The transformation was catalyzed by thermophilic Geobacillus stearothermophilus comb. nov., isolated from oil contaminated soil in Kuwait. Cholic acid, as the sole source of carbon, was added to G. stearothermophilus cells in phosphate buffer pH 7 and shaken at 65ºC for 5 days. Methodology: The cholic acid transformation products were extracted with ethyl acetate, purified on preparative TLC plates and their molecular structures were established from their spectral data. Results: The bacterium could selectively oxidize hydroxyl-groups at C3 and C7 while leaving the C12-hydroxyl-group unoxidized, in cholic acid. Five commonly found metabolites of cholic acid and a novel transformation product, 16α-hydroxycholic acid, were identified. Conclusion: Our results indicate that G. stearothermophilus can hydroxylate/oxidize a steroid nucleus at various ring positions, and has a unique ability for hydroxylation at C16α in cholic acid.

Evaluation of a Bacillus stearothermophilus tube test as a screening tool for anticoccidial residues in poultry

A Bacillus stearothermophilus var. calidolactis C953 tube test was evaluated for its ability in detecting the residue of selected anticoccidial drugs in poultry, specically sulfamethazine, furazolidone, and amprolium. Various concentrations of each drug were injected into chicken liver and kidney tissues and these tissues were tested to determine the drug detection limits for each drug. The detection limit was defined as the drug concentration at which 95% of the test results were interpreted as positive. The limits of detection in liver tissue were 0.35 microgram/ml for furazolidone, 0.70 microgram/ml for sulfamethazine and 7.80 microgram/ ml for amprolium. In kidney tissues, they were 0.30 microgram/ml for furazolidone, 0.54 microgram/ml for sulfamethazine, and 7.6 microgram/ml for amprolium. It was concluded that this tube test could be used to screen for the residue of these three drugs in poultry.

PROPERTIES OF ?-GALACTOSIDASE FROM BACILLUS STEAROTHERMOPHILUS / 微生物学通报

A themostable intracellular ? galactosidase from a thermophilic Bacillus stearothermophilus was purified by a combination of (NH 4) 2SO 4 fractionation,ion exchange (DEAE 22)and gel filtration (Sephades G 75).The optimum temperature and pH of the enzyme acivity were 60℃and pH6.4 respectively.The ? galatosidase activity exhibited thermosttability at 50 ℃.The enzyme was significaantly activated by alkali and alkali earth metal ions.The activity was inhibited by Zn 2+ 、 Fe 3+ 、 Cu 2+ Reducing agents enhanced ? galactosidase activity.Thiol binding agents drastically decreased the enzyme activity.The enzyme was specific for ? D glycosidic linkages,and the identity of the aglycone moiety also influenced enzyme activity.At 55℃the Km for O nitrophenyl ? D galactosidase(ONPG)and lactose were 2.63mmol/L and 4.39mmol/L, respectively,and Vmax for both substrates were 1.93?10 5 mmol.min 1 .mg 1 protein6.54?10 5 mmol.min 1 .mg 1 protein,respectively.The enzyme was inhibited by glucose (the products of lactose hydrolysis,ki 2.47mmol/L),but not by galactose.In addition,the enzyme possessed transgalactosylation activity.Galacto oligosaccharides,both tri and tetrasaccharide,were involved in the products during lactose hydrolysis.