| 1. |
- Ramchuran, Santosh, et al.
(författare)
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Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris
- 2006
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 71:4, s. 463-472
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Tidskriftsartikel (refereegranskat)abstract
- A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZ alpha B vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (alpha-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a "batch-induced" cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.
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| 2. |
- Vargas Calle, Virginia
(författare)
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Alkaliphilic Bacillus species and an alkaline active lipolytic enzyme
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Great Rift Valley running through Eastern Africa contains a large number of soda lakes. These unique lakes constitute among the most stable alkaline environments with a dense and varied population of microorganisms. Such microorganisms are invariable alkaliphiles and are considered to be a promising source of enzymes with unique features. Some enzymes produced by alkaliphilic microorganisms have found their way to industrial applications.Phylogenetic analysis and morphological and physiological characteristics of the isolates from a screening for lipase-producing microorganisms from a Kenyan alkaline soda lake showed some of them to be closely related to the Pseudomonas and Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining were Gram-positive, closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis, respectively. A new alkaliphilic Bacillus sp. was identified during the screening and based on detailed studies we proposed a novel alkaliphilic, halotolerant species of the genus Bacillus with the name Bacillus bogoriensis.Among the alkaliphilic Bacillus species isolated, Bacillus halodurans was selected due to its remarkable enzymes studied so far. A lipolytic enzyme was cloned and overexpressed in Escherichia coli. Production of the enzyme was mainly in the form of inclusion bodies from which recovery of the active enzyme was achieved. Since E. coli proved not to be a suitable host for production of soluble enzyme, Pichia pastoris was tested as an alternative host and the production of the heterologous enzyme in a soluble form was also studied. The enzyme was characterized as a phospholipase, also displaying hydrolysis of soluble and insoluble fatty substrates, and esterification of fatty acids.
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| 3. |
- Vargas Calle, Virginia, et al.
(författare)
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Bacillus bogoriensis sp nov., a novel alkallphilic, halotolerant bacterium isolated from a Kenyan soda lake
- 2005
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 55:2, s. 899-902
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Tidskriftsartikel (refereegranskat)abstract
- Strain LBB3(T) isolated from Bogoria soda lake in Kenya is an alkaliphilic, Gram-positive, strictly aerobic, non-motile, spore-forming bacterium. It was identified as a member of the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The organism grows optimally at 37 degrees C and pH 10. The G+C content of the genomic DNA is 37.5 mol%. 16S rRNA gene sequence analysis showed 95 and 96% sequence similarity with Bacillus pseudofirmus (DSM 8715(T)) and Bacillus alcalophilus (DSM 485(T)), respectively. Furthermore, DNA-DNA hybridization against these two Bacillus species showed 39.0 and 55.5% similarity, respectively. Based on our observations, strain LBB3(T) is proposed to represent a novel species of the genus Bacillus, for which the name Bacillus bogoriensis sp. nov. is proposed. The type strain of B. bogoriensis is LBB3(T) (= ATCC BAA-922(T) = LMG 22234(T)).
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| 4. |
- Vargas Calle, Virginia, et al.
(författare)
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Lipase-producing microorganisms from a Kenyan alkaline soda lake
- 2004
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Ingår i: Biotechnology Letters. - 1573-6776. ; 26:2, s. 81-86
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Tidskriftsartikel (refereegranskat)abstract
- Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55degreesC. Most of them showed optimal growth at 37degreesC and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.
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