Abstract

We identified from a collection of partially sequenced Aspergillus niger cDNA clones four cDNAs that were predicted to encode secreted lipases or phospholipases B/lysophospholipases. The deduced amino acid sequence of two of the four genes, lipA and lipB, exhibited approximately 50% identity with each other and with a lipase from the thermophilic fungus Thermomyces lanuginosus. The other two cDNAs, plbA and plbB, share amino acid sequence 63% identity with each other and about 65% identity with Aspergillus fumigatus PlbAp. We functionally expressed the four genes in Pichia pastoris and A. niger, and purified and characterized the recombinant proteins. LipA showed optimal activity at 40°C and pH 4.5-5.0. LipA was stable at a pH range of 2.2-10.6 and up to 70°C. The pH optimum for LipB was pH 3.5-4.0, and it was stable between pH 3.0 and pH 9.6. The temperature optimum for LipB was 15°C and it retained 70% of its peak activity at 0°C. LipB has the lowest temperature activity profile when compared with reported cold-adapted enzymes. Substrate specificity determination with triacylglycerols and p -nitrophenyl esters showed that both lipases preferred esters with middle- and long-chain fatty acids. The pH optimum for the lysophospholipase activity encoded by PlbA was 3.0 and the activity was stable during incubation from pH 2 to 8.6. The temperature optimum of PlbA was 50°C and it retained at least 80% activity when incubated at 50°C for 3 h. PlbA exhibited a rather broad specificity towards lysophospholipids with maximal activity on lysophosphatidy1choline and lysophosphatidylserine and preferred the shorter C12 substrate relative to substrates with 14 to 18 carbons. PlbA did not exhibit phospholipase B, A1 or A2, lipase or general esterase activity and appeared to be lysophospholipid specific. PlbA lysophospholipase activity displayed cooperative kinetics with a Hill coefficient of 2.036. Recombinant PlbB could only be expressed at an extremely low level in both Pichia pastoris and Aspergillus niger. We were unable to purify and characterize this enzyme