Biodegradation of lipids by wood sapstaining OPHIOSTOMA SPP (2009)

Canada is the largest exporter of softwood lumber in the world. Sapstain caused by fungi is a costly problem for the Canadian lumber industry. To maintain and increase future sales and exports of lumber requires not only effective, but also environmentally acceptable anti-sapstain methods. A thorough understanding of the physiological and biochemical features of sapstaining fungi should facilitate the development of new methods of protection. This work was carried out to determine the available lipid nutrients in wood and to understand the biodegradation mechanisms of lipids by sapstaining Ophiostoma species. Lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) constitutes the greatest volume of wood harvested in British Columbia. As with all wood species, the freshly sawn sapwood is susceptible to sapstain. The total extractives content in the sapwood at the breast height of a 35-year-old lodgepole pine tree was 1.9-2.2% of oven dry weight, which included 1.1-1.3% triglycerides (TG), 0.02-0.03% free fatty acids (FA), 0.3-0.4% free resin acids (RA), and 0.15- 0.21% steryl esters and waxes (SEAV). A considerably higher content of extractives was present in heartwood, i.e., 9.7-12.4%, including 0.6-0.7% TGs, 0.8-1.1% free FAs, 4.0-5.6% free RAs, and 0.25-0.33% SE/Ws. The wood TGs were composed mainly of oleic (18:1), linoleic (18:2), linolenic (18:3) and palmitic (16:0) acids. The dominant RA was palustric acid, followed by abietic, neoabietic, dehydroabietic, isopimaric, pimaric, and sandaracopimaric acids. The ability of three sapstaining fungal species, Ophiostoma piceae 387N, O. ainoae 701 A, and O. piliferum 55H to degrade and utilize the major lipids in the sapwood of lodgepole pine and trembling aspen (Populus tremuloides Michx.) was investigated. The fungal growth rate in wood was monitored by quantifying ergosterol extracted from colonized wood. After two weeks colonization, the TGs in wood were degraded by 50% to 80%, which resulted in an accumulation of free FAs in the wood. Lipases (glycerol ester hydrolases, EC 3.1.1.3) are the enzymes responsible for hydrolyzing TGs into glycerol and FAs which are assimilable by fungal cells. Extracellular lipase activity of O. piceae 387N was detected both in colonized wood and in liquid culture. The effect of various factors (carbon sources, nitrogen sources, and medium pH) on the growth and lipase activity of O. piceae 387N were examined in liquid culture. The extracellular lipase secretion was enhanced in the presence of triglycerides. The composition of a medium was optimized for a high extracellular lipase production, which contained 2% olive oil as a carbon source and 0.5% ammonium sulfate and 3% peptone as nitrogen sources with an initial medium pH of 5.0. A major extracellular lipase was purified from the liquid culture filtrates of O. piceae 387N by hydrophobic interaction chromatography and anion exchange chromatography. This lipase was characterized as a monomer with a molecular weight of 35 kDa, and was glycosylated, containing 10.1% carbohydrates. It was resolved as a single band on SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) gels, whereas 3 bands at pi's 4.3, 4.1 and 3.8 were observed on LEF (isoelectric focusing) gels. Lipolytic stain demonstrated that the three bands on IEF gels were lipolytically active. The 3 isoforms were found to have a same N - terminal sequence as D¹-V²-S³-V⁴-T⁵-T⁶-T⁷-D⁸-I⁹-D¹⁰-A¹¹-L¹²-A¹³-F¹⁴-F¹⁵-T¹⁶-Q¹⁷-W¹⁸-A¹⁹-G²⁰. The purified O. piceae 387N lipase was stable at pH's 4 to 8 and at temperatures below 40°C. The pH and temperature optima for activity were approximately pH 5.2 and 30°C, respectively. Enzyme activity was not influenced by N-ethylmaleimide, P-mercaptoethanol, and dithiothreitol, was slightly enhanced by Ca²⁺ and Mn²⁺, and was severely inhibited by Hg and Fe³⁺, diethyl pyrocarbonate, diethyl /?-nitrophenyl phosphate, butyric acid, caproic acid, and SDS. The lipase showed high specificity toward substrates with intermediate and long chain FA residues, and belonged to a group of 1(3) positional specific lipases. The rate of hydrolysis of the lipase toward a triglyceride (l,3-dipalmitoyl-2-oleoyl-glycerol) was 25-50 fold higher than that toward the waxes (oleyl esters) and cholesteryl esters. Finally, it was conclusively shown that the purified lipase could effectively release fatty acid residues from the triglycerides isolated from wood. The data and information obtained in this work have contributed to the understanding of the physiological and biochemical features of sapstaining Ophiostoma species. The large amounts of various lipids in wood, in particular TGs and FAs, are important carbon and energy sources for the sapstaining fungi, which are capable of secreting extracellular lipases to degrade TGs. The information implies that the growth of sapstaining fungi may be hindered by disrupting the metabolic processes of lipid utilization, or by applying biological competitors which are more efficient in assimilating lipids and other nutrients in wood.