An Organic Solvent-Tolerant Alkaline Lipase from Cold-Adapted Pseudomonas mandelii: Cloning, Expression, and Characterization

Junsung KIM, Sei-Heon JANG, ChangWoo LEE
2013 Bioscience, biotechnology and biochemistry  
A gene encoding a novel organic solvent-tolerant alkaline lipase, lipS (GenBank ID JQ071496), was cloned from cold-adapted Pseudomonas mandelii. Recombinant LipS was expressed in Escherichia coli as a 32-kDa soluble protein and was purified by standard procedures. It maintained more than 80% of its activity under alkaline conditions, pH 8-10.5, with an apparent optimum temperature range of 40-50 C. It maintained thermal stability from 4 to 50 C. After 1 h of incubation at 60 C, approximately
more » ... of its activity remained. It retained its activity in organic solvents, and activity increased in the presence of ethanol and of DMSO. Our data indicate that LipS is an alkaline lipase with relatively high thermal stability and notable tolerance of organic solvents. Key words: alkaline lipase; organic solvent; Pseudomonas mandelii; psychrotrophic bacterium Lipase (EC 3.1.1.3) belongs to the / hydrolase superfamily. It catalyzes the hydrolysis of triacylglycerols into glycerol and free fatty acids. 1) The active site of lipase possesses a catalytic triad consisting of serine, aspartate, and histidine residues. 1) Lipases from coldadapted bacteria have gained considerable attention in biotechnological applications due to their high catalytic activity at low temperature. 2) To function in lowtemperature environments, cold-adapted lipases have structures that increase their flexibility relative to higher-temperature lipases, in a selected area or in overall protein structure. The differences include weakened intramolecular hydrogen bonds and salt bridges, reduced internal hydrophobic interactions, and loop extension. 2,3) However, these features have also rendered cold-adapted lipases more susceptible than their warmer-temperature counterparts to denaturation by heat, extreme pH, and organic solvents. Since the identification of the first organic solventtolerant lipase, derived from Pseudomonas aeruginosa LST-03, 4) several lipases tolerant of organic solvent have been cloned and characterized, the majority originating in Pseudomonas sp. or Bacillus sp. 5,6) Organic solvent-tolerant lipases from cold-adapted bacteria have notable potential as catalysts for the organic synthesis of chiral intermediates at low temperatures. 2) Only a few organic solvent-tolerant lipases from cold-adapted bacteria have been reported. [7] [8] [9] Here, we report the cloning, expression, and characterization of an organic solvent-tolerant alkaline lipase, LipS (GenBank ID JQ071496), from Pseudomonas mandelii JR-1. P. mandelii is a cold-adapted bacterium that grows at 4 C but not at 37 C, with an apparent optimum temperature range of 25-30 C. 10,11) To produce a clone, we utilized the genome sequence of Pseudomonas fluorescens Pf0-1, 12) which is closely related to P. mandelii, from among completed microbial genomes. The lipS gene was cloned using primers based on the non-coding region sequences surrounding the P. fluorescens Pf0-1 lipase gene (UniProt ID Q3KIU1). LipS showed high sequence similarity to organic solvent-tolerant lipase rPFL from Pseudomonas fluorescens JCM5963 13) (80% sequence identity) and to Lip9 from Pseudomonas aeruginosa LST-03 14,15) (44% sequence identity). We found that LipS is an alkaline lipase that demonstrates more than 80% of its activity at pH 8-10.5, is tolerant of organic solvents, and has relatively high thermal stability. Materials and Methods Materials. Bacterium Pseudomonas mandelii JR-1 was deposited in the Korean Collection for Type Culture (Daejeon, Korea) as KCTC 12151BP. TA cloning vector was purchased from iNtRON Biotechnology (Daejeon, Korea). pET28a expression vector was from Novagen (Boston, MA), Ex Taq DNA polymerase was from Takara Biotechnology (Seoul, Korea), HisTrap FF column and Hirap Q-Sepharose FF column were from GE Healthcare (Piscataway, NJ), esters for p-nitrophenyl were from Sigma (St. Louis, MO), and all other reagents were from Sigma unless noted otherwise. Gene cloning of lipS. The lipS gene was cloned from Pseudomonas mandelii JR-1 by polymerase chain reaction (PCR) in two steps. At the first step, primers were designed based on non-coding sequences surrounding the lipase coding sequence (UniProt ID Q3KIU1) in the Pseudomonas fluorescens Pf0-1 genome. The forward primer (GS4F) was 5 0 -GTGTTTCTCGGTTTCGCTCT-3 0 , approximately 170 bp upstream of Q3KIU1, and the reverse primer (GS5R) was 5 0 -GTGA-TTCTTTCCGAGCGCTG-3 0 , approximately 40 bp downstream. The resulting PCR product from P. mandelii JR-1 was subcloned into a TA vector and then sequenced. At the second step, the lipS gene was amplified from the TA vector and subcloned into a pET28a vector. The forward primer used (GS56F) was 5 0 -GAGAGACATATGATGTCG-CAAGGTTCTGCCACG-3 0 (Nde I site underlined and the Nterminal part of LipS in bold-face type). The reverse primer used (GS57R) was 5 0 -GAGAGACTCGAGTTACACGCCAGCCCCTTT-CAATC-3 0 (Xho I site underlined and the C-terminal part of LipS in bold-face type). A His 6 and an 11 amino acid linker sequence (HHHHHHSSGLVPRGSHM), which came from a pET28 vector, was located on the N-terminus of LipS. The construct was confirmed by DNA sequencing.
doi:10.1271/bbb.120733 pmid:23391923 fatcat:7377ag2mr5eirpwn4g5dchjbsm