Producción de plásticos biodegradables a partir de bacterias de hábitats salinos aisladas de la Laguna de Ayarza / Production of biodegradable plastics from saline habitats bacteria isolated from Laguna de Ayarza

La contaminación por plásticos petroquímicos es una grave amenaza para el medio ambiente que requiere im-plementar alternativas como los bioplásticos para lograr un desarrollo sostenible. Los polihidroxialcanoatos (PHA) son polímeros utilizados para la producción de plásticos biodegradables y que han llamado la atención como sustitutos de los plásticos de base fósil. Sin embargo, el costo de producción de los PHA constituye una barrera para su producción industrial a gran escala. Las de bacterias de hábitats salinos son microorganismos prometedores para la síntesis de PHA debido a sus características tales como altos requisitos de salinidad que previenen la contaminación microbiana, la alta presión osmótica intracelular que permite una fácil lisis celular para purificar los PHA y la capacidad para usar un amplio espectro de sustratos. La presente investigación planteó determinar las cepas nativas de bacterias halófilas y halotolerantes de la Laguna de Ayarza capaces de producir PHA, establecer la capacidad que tienen de utilizar residuos agrícolas para la producción de PHA y determinar su eficiencia. Esto se logró a través de la inoculación de las cepas productoras de PHA en medios de fermentación con pulpa de café, cáscaras de plátanos y salvado de trigo lo que permitió determinar las cepas más eficientes. Se encontró que las bacterias productoras de PHA pertenecen a las especies: Alcaligenes faecalis, Bacillus idriensis, Bacillus megaterium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis y Sta-phylococcus capitis y que las cepas AP21-14, AP21-10 y AP21-03 mostraron los mejores resultados que podrían ser prometedores para la producción a nivel industrial.

Pollution by petrochemical plastics is a serious threat to the environment that requires the implementation of al-ternatives such as bioplastics to achieve sustainable development. Polyhydroxyalkanoates (PHAs) are polymers used for the production of biodegradable plastics and have drawn attention as substitutes for fossil-based plastics. However, the cost of producing PHAs constitutes a barrier to their large-scale industrial production. Bacteria from saline environments bacteria are promising microorganisms for PHA synthesis due to their characteristics such as high salinity requirements that prevent microbial contamination, high intracellular osmotic pressure that allows easy cell lysis to purify PHAs, and the ability to use a broad spectrum of substrates. This research project aimed to determine the native strains of halophilic and halotolerant bacteria from Laguna de Ayarza capable of producing PHA, establish their ability to use agricultural residues for the production of PHA, and determine their efficiency. This was achieved through the inoculation of the PHA-producing strains in fermentation media with coffee pulp, banana peels and wheat bran, which allowed determining the most efficient strains. It was found that the PHA-producing bacteria belong to the species: Alcaligenes faecalis, Bacillus idriensis, Bacillus mega-terium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis and Staphylococcus capitis and that the strains AP21-14, AP21-10 and AP21-03 showed the best results that could be promising for production at an industrial level.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens Against Aspergillus and Penicillium spp. Predominant in Stored Rice Grains: Study II

In our previous studies, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15 have been shown to be antagonistic to Aspergillus flavus in stored rice grains. In this study, the biocontrol activities of these strains were evaluated against Aspergillus candidus, Aspergillus fumigatus, Penicillium fellutanum, and Penicillium islandicum, which are predominant in stored rice grains. In vitro and in vivo antifungal activities of the bacterial strains were evaluated against the fungi on media and rice grains, respectively. The antifungal activities of the volatiles produced by the strains against fungal development and population were also tested using I-plates. In in vitro tests, the strains produced secondary metabolites capable of reducing conidial germination, germ-tube elongation, and mycelial growth of all the tested fungi. In in vivo tests, the strains significantly inhibited the fungal growth in rice grains. Additionally, in I-plate tests, strains KU143 and AS15 produced volatiles that significantly inhibited not only mycelial growth, sporulation, and conidial germination of the fungi on media but also fungal populations on rice grains. GC-MS analysis of the volatiles by strains KU143 and AS15 identified 12 and 17 compounds, respectively. Among these, the antifungal compound, 5-methyl-2-phenyl-1H-indole, was produced by strain KU143 and the antimicrobial compounds, 2-butyl 1-octanal, dimethyl disulfide, 2-isopropyl-5-methyl-1-heptanol, and 4-trifluoroacetoxyhexadecane, were produced by strain AS15. These results suggest that the tested strains producing extracellular metabolites and/or volatiles may have a broad spectrum of antifungal activities against the grain fungi. In particular, B. megaterium KU143 and P. protegens AS15 may be potential biocontrol agents against Aspergillus and Penicillium spp. during rice grain storage.

Compatibility of Azospirillum brasilense and Pseudomonas fluorescens in growth promotion of groundnut ( Arachis hypogea L)

ABSTRACT We attempted to study the compatibility among plant beneficial bacteria in the culture level by growing them near in the nutrient agar plates. Among all the bacteria tested, Rhizobium was found to inhibit the growth of other bacteria. From the compatible group of PGPR, we have selected one biofertilizer (Azospirillum brasilense strain TNAU) and one biocontrol agent (Pseudomonas fluorescens strain PF1) for further studies in the pot culture. We have also developed a bioformulation which is talc powder based, for individual bacteria and mixed culture. This formulation was used as seed treatment, soil application, seedling root dip and foliar spray in groundnut crop in vitro germination conditions. A. brasilense was found to enhance the tap root growth and P. fluorescens, the lateral root growth. The other growth parameters like shoot growth, number of leaves were enhanced by the combination of both of the bacteria than their individual formulations. Among the method of application tested in our study, soil application was found to be the best in yielding better results of plant growth promotion.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens against Aspergillus flavus and Aflatoxin Production on Stored Rice Grains

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.

Screening of alginate lyase-producing bacteria and optimization of media compositions for extracellular alginate lyase production

Background: Alginate is a linear polysaccharide consisting of guluronate [polyG] and mannuronate [polyM] subunits

Methods: In the initial screening of alginate-degrading bacteria from soil, 10 isolates were able to grow on minimal medium containing alginate. The optimization of cell growth and alginate lyase [algL] production was carried out by the addition of 0.8% alginate and 0.2-0.3 M NaCl to the culture medium. Of 10 isolates, one was selected based on its fast growth rate on minimal 9 medium containing 0.4% sodium alginate. The selected bacterium, identified based on morphological and biochemical characteristics, as well as 16S rDNA sequence data, was confirmed to be an isolate belonging to the genus Bacillus and designated as Bacillus sp. TAG8

Results: The results showed the ability of Bacillus sp. TAG8 in utilizing alginate as a sole carbon source. Bacillus sp. TAG8 growth and algL production were augmented with an increase in sodium alginate concentration and also by the addition of 0.2-0.3 M NaCl. Molecular analysis of TAG8 algL gene showed 99% sequence identity with algL of Pseudomonas aeruginosa PAO1. The algL produced by Bacillus sp. TAG8 cleaved both polyM and polyG blocks in alginate molecule, as well as acetylated alginate residues, confirming the bifunctionality of the isolated lyase

Conclusion: The identification of novel algL genes from microbial communities constitutes a new approach for exploring lyases with specific activity against bacterial alginates and may thus contribute to the eradication of persistent biofilms from clinical samples

Dissolving mechanism of strain P17 on insoluble phosphorus of yellow-brown soil

Strain P17 was a bacterial strain identified as Bacillus megaterium isolated from ground accumulating phosphate rock powder. The fermentation broth of strain P17 and the yellow-brown soil from Nanjing Agricultural University garden were collected to conduct this study. The simulation of fixed insoluble phosphorous forms after applying calcium superphosphate into yellow-brown soil was performed in pots, while available P and total P of soil were extremely positive correlative with those of groundwater. Then the dissolving effect of strain P17 on insoluble P of yellow-brown soil was studied. Results showed that Bacillus megaterium strain P17 had notable solubilizing effect on insoluble phosphates formed when too much water-soluble phosphorous fertilizer used. During 100 days after inoculation, strain P17 was dominant. Until the 120th day, compared with water addition, available P of strain P17 inoculation treated soil increased by 3 times with calcium superphosphate addition. Besides available P, pH, activity of acid and alkaline phosphatase and population of P-solubilizing microbes were detected respectively. P-solubilizing mechanism of P-solubilizing bacteria strain P17 seems to be a synergetic effect of pH decrease, organic acids, phosphatase, etc.

Bioconversion of cassava starch by-product into bacillus and related bacteria polyhydroxyalkanoates

Background: Unlike petroleum-based synthetic plastics, biodegradable biopolymer generation from industrial residue is a key strategy to reduce costs in the production process, as well as in the waste management, since efficient industrial wastewater treatment could be costly. In this context, the present work describes the prospection and use of bacterial strains capable to bioconvert cassava starch by-product into biodegradable polyhydroxyalkanoates (PHAs). Results: The first step of this study was the bacterial competence screening which was conducted with 72 strains covering 21 Bacillus and related species. The microorganism growth in a medium with a starch substrate was measured by an innovative MTT assay, while the ability of the bacteria to secrete amylase and produce PHA was evaluated by the Nile Red Dye method. Based on growth and potential for PHA production, four isolates were selected and identified as Bacillus megaterium by 16S rRNA sequencing. When cultivated in hydrolyzed cassava starch by-product, maximum production reached 4.97 g dry biomass/L with 29.7 percent of Poly-(3-hydroxybutyrate) (characterized by FTIR). Conclusions: MTT assay proved to be a reliable methodology for monitoring bacterial growth in insoluble media. Selected amylolytic strains could be used as an alternative industrial process for biodegradable plastics production from starchy residues, reducing costs for biodegradable biopolymer production and wastewater treatment operations.

Recombinant expression and refolding of the c-type lysozyme from Spodoptera litura in E. coli

The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95 percent purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli.

The nematicidal effect of some bacterial biofertilizers on Meloidogyne incognita in sandy soil

In a greenhouse experiment, the nematicidal effect of some bacterial biofertilizers including the nitrogen fixing bacteria (NFB) Paenibacillus polymyxa (four strains), the phosphate solubilizing bacteria (PSB) Bacillus megaterium (three strains) and the potassium solubilizing bacteria (KSB) B. circulans (three strains) were evaluated individually on tomato plants infested with the root-knot nematode Meloidogyne incognita in potted sandy soil. Comparing with the uninoculated nematode-infested control, the inoculation with P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2, increased the counts of total bacteria and total bacterial spores in plants potted soil from 1.2 to 2.6 folds estimated 60 days post-inoculation. Consequently, the inoculation with P. polymyxa NFB7 increased significantly the shoot length (cm), number of leaves / plant, shoot dry weight (g) / plant and root dry weight (g) / plant by 32.6 percent, 30.8 percent, 70.3 percent and 14.2 percent, respectively. Generally, the majority treatments significantly reduced the nematode multiplication which was more obvious after 60 days of inoculation. Among the applied strains, P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 inoculations resulted in the highest reduction in nematode population comparing with the uninoculated nematode-infested control. They recorded the highest reduction in numbers of hatched juveniles/root by 95.8 percent, females/root by 63.75 percent and juveniles/1kg soil by 57.8 percent. These results indicated that these bacterial biofertilizers are promising double purpose microorganisms for mobilizing of soil nutrients (nitrogen, phosphate and potassium) and for the biological control of M. incognita.

Preliminary antimicrobial activity and cytotoxicity of leaf extracts of mesua nagassarium (Burm.f. ) / Actividad antimicrobiana preliminar y citotoxicidad de extractos de hojas de Mesua nagassarium (Burm.f. )

In the present study the in vitro antimicrobial activity, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), of different extracts of leaves of Mesua nagassarium were evaluated against 13 pathogenic microorganisms. The methanol extract and its pet-ether and carbon tetrachloride soluble fractions showed the highest antimicrobial activity. The carbon tetrachloride soluble fraction showed the maximum inhibition zone of 24.33 mm against Bacillus megaterium with MIC and MBC values of 7.81 ug/ml and 250 ug/ml, respectively. Ciprofloxacin (30 ug/disc) was used as standard antimicrobial agent. In the Brine shrimp lethality bioassay, the crude methanol extract and its carbon tetrachloride soluble fraction showed significant cytotoxicity with LC50 of 2.99 and 1.74 ug/ml, respectively as compared vincristine sulphate (LC50 value 0.543 ug/ml).

En el presente estudio se evaluó la actividad antimicrobiana in vitro, incluyendo la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM), de diferentes extractos obtenidos de hojas de Mesua nagassarium en 13 microorganismos patógenos. El extracto metanólico y sus fracciones solubles en éter de petróleo y tetracloruro de carbono, mostraron la mayor actividad antimicrobiana. La fracción de compuestos solubles en tetracloruro de carbono mostró la zona de inhibición máxima de 24.33 mm en Bacillus megaterium con valores de CIM y and CBM de 7.81 ug/ml y 250 ug/ml, respectivamente. Como agente antimicrobiano estándar se utilizó ciprofloxacina (30 ug/disco). En el bioensayo de mortalidad de Brine shrimp el extracto metanólico y su fracción soluble en tetracloruro de carbono mostraron importante citotoxicidad con CL50 de 2.99 y 1.74 ug/ml, respectivamente, comparadas con el sulfato de vincristina (CL50 0.543 ug/ml).

Production of polyhydroxyalkanoate [PHAs] and copolymer [P[HB-co-HV]] by soil bacterial isolates in batch and two-stage batch cultures

NINETY TWO local bacterial isolates, from the rhizosphere and soil around the root system of bean [Viciafaba] grown in Kalubia Governorate in Egypt, were bio-prospected for polyhydroxyalkanoate [PHA] accumulation. Three isolates accumulated >/=20% of PHAs, they were identified as Pseudomonas flu orescens S48, Bacillus megaterium 7A and B. megaterium UBFI9. The tested isolates gave the maximum PHAs content on basal medium containing glucose and ammonium sulfate at C/N ratio of 30/1 after 72 hr at 30°C using shake flask culture technique. Two-stage batch were implemented with varying loading levels of nitrogen and phosphorus, inoculated with washed cells. Nitrogen omission of 70% led to increase the PHAs content by 19%, 3% and 8.5% using washed cells of Ps. fluorescens S48, B. megaterium UBF 19 and Bacillus megaterium 7A, respectively comparing with batch production on the same medium after 72 hr. The Copolymer poly[hydroxybutyrate-co-hydroxyvalerate] [P [HB-co-HV]] content level was increased when valerie/glucose [V/G] was 0.19 mol.mo[-1] after 96 hr being 25.97% and 20.11% by Ps. fluorescens S48 and B. megaterium UBFI9, respectively and reached 23.73% by B. megaterium 7A at propionic/glucose [PIG] of 0.5 mol.mol[-1]. The corresponding highest values of valeric content of copolymer at V/G 3.08 mol.mol[-1] were 63%, 49% and 45%, respectively, comparing with other V/G ratios by using GC analysis . Replacing glucose with 2% corn oil or 1% soybean oil increased the PHAs content of Ps. fluorescens S48 cells to 54.21% and 52.12%, respectively, after 72

Production conditions of exopolysaccharide from bacillus megaterium identified by 16S rRNA gene sequencing

THE BACILLUS megaterium exopolysaccharide [EPS] producer was isolated from the Egyptian soil and identified by molecular biology technique [16S rRNA]. It was found that the exopoiysaccharide showed a high yield after 72 hr, with an initial pH of 5.0, in 250 ml Erlenmeyer flasks containing 50 ml of culture, medium having 2% sucrose as a sole carbon source. The yeast extract [0.1%] and animonium chloride [0.1%] were the best organic and inorganic nitrogen source, respectively, as they gave the maximum EPS production. Optimum incubation temperature was found to be 35°C. Pretreated molasses from sugarcane and sugarbeet were used as carbon source and they gave a considerable yield [-tow fold] in relation to sucrose. The pretreatment of molasses by sulfuric acid showed high yield of polysaccharide and maximum yield coefficient

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey / Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.

Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por PCR secuencias de ADN de los genes nhe (A, B y C), HBL (A, B, C, D), cytK y bceT. La relación entre las variables fue evaluada mediante un análisis de componentes principales, donde se encontró que los aislamientos de B. cereus mostraron una correlación positiva entre actividad de coagulasa (coagulasa libre) y presencia de los genes del complejo HBL y bceT, mientras que en B. megaterium se halló una alta correlación positiva entre actividad de coagulasa (libre y fija) y actividad hemolítica, pero no se observó correlación significativa entre la presencia de genes de virulencia y dichas actividades. Este estudio constituye el primer registro de la presencia de los genes cyt K y NHE en cepas de B. megaterium y el primer trabajo que analiza la relación entre la actividad de coagulasa y la presencia de genes de virulencia en B. cereus y B. megaterium. La presencia del gen cytK en ambas especies resultó totalmente independiente del resto de los factores de virulencia analizados. Nuestros hallazgos sugieren que la miel podría vehiculizar enfermedades transmisibles por alimentos debido a la presencia de cepas de B. cereus y B. megaterium potencialmente tóxicas.

Enhancement of 2-keto-L-gulonic acid production using three-stage pH control strategy / 生物工程学报

The aim of this study was to improve the 2-keto-L-gulonic acid (2-KLG) production efficiency by Ketogulonicigenium vulgare and Bacillus megaterium by using multi-stage pH control strategy. The effect of pH on the cell growths and 2-KLG production showed that the optimum pH for K. vulgare and B. megaterium cell growth were 6.0 and 8.0, respectively, while the optimum pH for 2-KLG production was 7.0. Based on the above results, we developed a three-stage pH control strategy: the pH was kept at 8.0 during the first 8 h, then decreased to 6.0 for the following 12 h, and maintained at 7.0 to the end of fermentation. With this strategy, the titer, productivity of 2-KLG and L-sorbose consumption rate were achieved at 77.3 g/L, 1.38 g/(L x h) and 1.42 g/(L x h), respectively, which were 9.7%, 33.2% and 25.7% higher than the corresponding values of the single pH (pH 7.0) control model.

Enhancing 2-keto-L-gulonic acid production under hyperosmotic stress by adding sucrose / 生物工程学报

This study aimed to further enhance 2-keto-L-gulonic acid (2-KLG) production efficiency. A strategy for enhancing Ketogulonigenium vulgare growth and 2-KLG production by improving B. megaterium growth with sucrose was developed based on the time course of osmolality during 2-KLG industrial scale fermentation and effects of osmolality on cells growth and 2-KLG production. Results showed that the accumulation of 2-KLG and the feeding of alkaline matter led to an osmolality rise of 832 mOsmol/kg in the culture broth. High osmotic stress (1 250 mOsmol/kg) made the growth ofB. megaterium and K. vulgare decreased 15.4% and 31.7%, respectively, and consequently the titer and productivity of 2-KLG reduced 67.5% and 69.3%, respectively. When supplement sucrose under high osmotic condition (1 250 mOsmol/kg), B. megaterium growth was significantly improved, with the result that 2-KLG production was increased 87%. Furthermore, by applying this sucrose addition strategy further to batch fermentation in 3 L fermentor, the productivity of 2-KLG increased 10.4%, and the duration of fermentation declined 10.8%. The results presented here provide a potential strategy for enhancing the target metabolites produced by mixed strains at environmental stress.

Cloning and expression of lipase gene to enantioselective resolution of (S)-ketoprofen / 生物工程学报

We screened a strain NK13 for a certain extent asymmetric hydrolysis the rac-ketoprofen Chloroethyl ester to (S)-Ketoprofen. As identified, NK13 was Bacillus megaterium. Digested NK13 genomic DNA with Sau3AI partially and recovered the fragment from 2 kb to 6 kb, cleaved the plasmid of pUC18 with BamH I, ligated the 2-6 kb fragment of NK13 genomic DNA into pUC18 plasmid, and then transformed an Escherichia coli strain DH5alpha. We created the gene library of NK13 and obtained a positive clone, pUC-NK1 in the library from the tributyrin flat. The result of sequencing showed that there was a whole open read frame (ORF) of 633 bp lipase gene in the plasmid of pUC-NK1. To compare with the genes of GenBank, this lipase gene was reported firstly (GenBank Accession No. EU381317). The lipase gene was amplified by PCR, using pUC-NK1 plasmid as template, and subcloned into the high expression vector pET21b(+) under the control of T7 promoter. The recombinant plasmid, pET-NKest1, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant lipase protein. After 3 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), lipase was expressed. SDS-PAGE analysis showed that the relative molecular mass of the lipase protein was about 20 kDa. The result of high performance liquid chromatography (HPLC) showed that the conversion rate of the recombinant strain was fifty times than the wild strain NK13's. The (S)-Ketoprofen enantiomeric excess of the recombinant strain was 75.28%, which indicated that the lipase could hydrolyze (S)-Ketoprofen Chloroethyl ester firstly. If we research the conditions of the hydrolysis rac-ketoprofen Chloroethyl ester of this lipase further, maybe it could offer a foundation to product (S)-Ketoprofen industrially.

Effect of carbon source and precursors on the production of plant growth regulators by azotobacter chroococcum [R19] and bacillus megaterium var. phosphaticum [R44]

An experiment was carried out to examine the effect of carbon source, tryptophan and adenine concentrations on the production of growth regulators by A. chroococcum [R19] and B. megaterium var. phosphaticum [R44]. Mannitol and glucose were the best carbon sources for the production of plant growth regulators [PGRs] by A. chroococcum and B. megaterium var. phosphaticum, respectively. A. chroococcum produced higher amounts of zeatin and kinetin compared to those produced by B. megaterium var. phosphaticum while B. megaterium var. phosphaticum produced higher amounts of [9R] benzyl adenine and [9G] bcnzyl adenine compared with those produced by A. chroococcum. Production of auxins, gibberellic acid [GA[3]] and cytokinins was increased with increasing tryptophan concentration. The highest amounts of PGRs produced by the two strains were obtained with tryptophan at 1000 micro M. Highest amounts of PGRs produced by A. chroococcum and B. megaterium var. phosphaticum at 10 and 100 micro M of adenine, respectively. Also, the produced amounts of gibberellic acid and cytokinin than that produced by B. megaterium var. phosphaticum. Generally, obtained data showed that the application of the optimal conditions together gave highest amounts of PGRs as compared with the other individual factors. This result is logic and was anticipated

Cloning, expression and the characterization of beta-amylase from a Bacillus megaterium WS06 / 生物工程学报

A beta-amylase gene (amyG) was cloned from a Bacillus megaterium WS06 and expressed in the Escherichia coli. Nucleotide sequence anlysis showed the amyG gene is composed of 1638 bp (545 amino acid residues with a Mr of 60.194 kD). The AmyG shows 94.5% sequence homologies with beta-amylase from Bacillus megaterium DSM319 and presents a normal beta-amylase primary structure, constituted by three parts: the N-terminal signal sequence, the catalytic domain and the C-terminal starch binding domains. The deduced amino acid sequence revealed that several highly conserved regions of the glycosylhydrolase family 14. The amyG gene was overexpressed using the pET21a vector and Escherichia coli BL21(DE3). The recombinant enzyme was purified 7.4 fold to electrophoretic homogeneity and had a Mr of 57 kD (by SDS-PAGE). The enzyme was optimally active at pH 7.0 and 60 degrees C and showed stability at the temperature below 60 degrees C. This enzyme efficiently hydrolyzed starch to yield maltose from non-reducing chain ends by exo-cleavage mode.

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process / Exploración del uso de agentes antimicrobianos naturales y de campos eléctricos pulsantes para el control de bacterias contaminantes durante el proceso de elaboración de cerveza

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 °C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Diferentes antimicrobianos naturales disminuyeron la viabilidad de bacterias contaminantes aisladas en etapas críticas del proceso de producción de cerveza. En un extracto de malta, el agregado de 1 mg/ml de quitosano y de 0,3 mg ml de lúpulo permitió reducir la viabilidad de Escherichia coli a 0,7 y 0,1%, respectivamente, al cabo de 2 horas de incubación a 4 °C. El agregado de 0,0002 mg/ml de nisina, 0,1 mg/ml de quitosano o de 0,3 mg/ml de lúpulo inhibió selectivamente (10.000 veces más) el crecimiento de Pediococcus sp. respecto de la levadura de cerveza en un cultivo mixto. El agregado de 0,1 mg/ml de quitosano permitió disminuir la viabilidad de una cepa bacteriana termorresistente, Bacillus megaterium, hasta niveles no detectables. Por otra parte, el agregado de nisina, quitosano y lúpulo aumentó la estabilidad microbiológica durante el almacenamiento de cervezas inoculadas con Lactobacillus plantarum y Pediococcus sp. aislados de mosto de cerveza. La aplicación de campos eléctricos pulsantes (CEP) (3 pulsos de 8kV/cm) aumentó el efecto antimicrobiano de la nisina y del lúpulo, pero no el del quitosano. Los resultados obtenidos indicarían que el uso de antimicrobianos naturales en forma individual o en combinación con CEP puede constituir un procedimiento efectivo para el control de la contaminación bacteriana durante el proceso de elaboración y almacenamiento de la cerveza.

Optimization of fermentation conditions for P450 BM-3 monooxygenase production by hybrid design methodology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl(3), induction at OD(578) (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90x10(-3) U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl(3), inoculum concentration of 2.10%, induction at OD(578) equal to 1.07, and with 6.05 h of induction.