A novel lipase gene (lipB52) was isolated directly from the genomic DNA of Pseudomonas fluorescens B52 with the genome-walking method, an effective method for isolating lipase gene from bacteria. There was an open reading frame (ORF) of 1854 bp, which encoded 617 amino acids. The lipase gene (lipB52) was cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant Pichia pastoris were screened with a high throughput method. The recombinant was induced by methanol to secrete active lipase into the culture medium. The recombinant lipase LipB52 was also purified and characterized. The optimum temperature for the purified lipase LipB52 was 40 degrees C at pH 8.0. It exhibited better thermostability and pH stability than its homologs.