Identification and characterization of three novel lipases belonging to families II and V from Anaerovibrio lipolyticus 5ST.
Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14).
Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Ceredigion, UK.
Florence
F
Privé
Naheed N
NN
Kaderbhai
Susan
S
Girdwood
Hilary J
HJ
Worgan
Eric
E
Pinloche
Nigel D
ND
Scollan
Sharon A
SA
Huws
C Jamie
CJ
Newbold
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008066:Lipolysis; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D045854:Veillonellaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D008049:Lipase