Gene cloning, expression and characterization of two cold-adapted lipases from Penicillium sp. XMZ-9 / 生物工程学报
Chinese Journal of Biotechnology
; (12): 488-497, 2012.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-342468
Biblioteca responsável:
WPRO
ABSTRACT
Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry.
Texto completo:
Disponível
Contexto em Saúde:
Doenças Negligenciadas
Problema de saúde:
Doenças Negligenciadas
/
Zoonoses
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Penicillium
/
Filogenia
/
Proteínas Recombinantes
/
RNA Ribossômico 18S
/
Sequência de Bases
/
Reação em Cadeia da Polimerase
/
Fases de Leitura Aberta
/
Clonagem Molecular
/
Temperatura Baixa
/
Escherichia coli
Tipo de estudo:
Estudo prognóstico
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2012
Tipo de documento:
Artigo