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Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes

Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude... Annals of Microbiology, 57 (4) 495-501 (2007) Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes Lin WANG, Zhenming CHI*, Xianghong WANG, Zhiqiang LIU, Jing LI Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China Received 15 May 2007 / Accepted 27 September 2007 Abstract - Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After lipase activity of the yeast cultures was estimated, we found that nine yeast strains obtained in this study grown in the medium with olive oil could produce lipase. The results of routine identification and molecular methods show that they belonged to Candida intermedia YA01a, Pichia guilliermondii N12c, Candida parapsilosis 3eA2, Lodderomyces elongisporus YF12c, Candida quercitrusa JHSb, Candia rugosa wl8, Yarrowia lipolytica N9a, Rhodotorula mucilaginosa L10-2 and Aureobasidium pullulans HN2.3, respectively. The optimal pHs and temperatures of lipases produced by them were between 6.0 and 8.5 and between 35 and 40 ºC, respectively. Majority of lipases from the yeast strains were cell-bound and only lipase from A. pullulans HN2.3 was extracellular. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry. Key words: lipase, marine-derived yeasts, diversity, marine environments, lipid hydrolysis. INTRODUCTION been purified and characterised and the genes encoding lipase in Candida, Geotrichum, Trichosporon and Y. lipolyt- Lipases are a class of hydrolyases that are primarily ica have been cloned and overexpressed (Vakhlu and Kour, responsible for the hydrolysis and the synthesis of esters 2006; Sharma et al., 2001). It has been found that most of formed from glycerol and long-chain fatty acids in vitro and lipases are serine hydrolyases according to their biochemi- in vivo. They catalyse a wide range of reactions, including cal properties (Vakhlu and Kour, 2006). Although lipases hydrolysis, inter-esterification, alcoholysis, acidolysis, from Candida rugosa and Candida antarctica have been esterification and aminolysis. Therefore, lipases, especially extensively used in flavour industry, synthesis of lipophillic microbial lipases have many industrial applications in the antioxidants, detergent industry, lipid hydrolysis, biosen- detergent, food, flavour industry, biocatalytic resolution of sors and clinical purpose, very few studies exist on the pharmaceuticals, esters and amino acid derivatives, mak- lipase produced by the yeasts obtained from marine envi- ing of fine chemicals, agrochemicals, use as biosensor, ronments (Chi et al., 2006). bioremediation and cosmetics and perfumery (Hasan et al., After we screened over 400 yeast strains from different 2006). Following proteases and carbohydrases, lipases are marine environments, we found that some yeast strains considered to be the third largest group based on total from different marine environments could produce lipase. sales volume. The commercial use of lipases is a billion-dol- The main purpose of the present study was to analyse lar business that comprises a wide variety of different appli- diversity of lipase-producing yeasts from different marine cations. Usually, extracellular lipase is produced by sub- environments. We also carried out the hydrolysis of various merged liquid fermentation, solid state fermentation and oils by the crude lipases or cell-bound lipases produced by immobilised cell culture (Sharma et al., 2001). Numerous the lipase-producing yeasts. To our knowledge, this is the species of bacteria, yeasts and molds were found to pro- first report on the lipase-producing yeasts derived from the duce lipases. Among the terrestrial yeasts, several Candida marine environments. spp., Yarrowia lipolytica, several Rhodotorula spp., some Pichia spp., Saccharomycopsis crataegensis, Torulospora globosa and Trichosporon asteroides have been found to be MATERIALS AND METHODS able to produce lipase (Vakhlu and Kour, 2006; Sharma et al., 2001). Because most of yeast strains are considered as Sampling. Different samples of seawater and sediments non-pathogenic, the processes for lipase production based in China South Sea, China East Sea, Indian Ocean and the on yeasts have been classified as GRAS (generally regard- Pacific Ocean were collected during the Antarctic explo- ed as safe). Extracellular lipases from several yeasts have ration in 2004 and hypersaline sea water, sediments of the salterns, different species of marine animals and algae along the coast of Qingdao, China were also col- * Corresponding author. Phone and Fax: 0086-532-82032266; lected. E-mail: zhenming@sdu.edu.cn 496 L. Wang et al. Isolation of marine yeasts. Two millilitres of the seawa- primer was NL-4 (5’-GGTCCGTGTTTCAAGACGG-3’) (Sugita ter or 2 g of the sediments or 2 ml of homogenised guts of et al., 2003). The reaction system (25 µl) was composed of marine animals or homogenised marine algae were sus- 10x buffer 2.5 µl, dNTP 0.8 µM, MgCl 1.5 mM, NL-1 0.5 pended in 20 ml of YPD medium containing 2% (w/v) glu- µM, NL-4 0.5 µM, Taq DNA polymerase 1.25 U, template cose, 2% (w/v) polypeptone and 1% (w/v) yeast extract DNA 1.0 µl and H O 16.6 µl. The conditions for the PCR and supplemented with 0.05% (w/v) chloramphenicol amplification were as follows: initial denaturation at 94 °C immediately after sampling and cultivated at natural tem- for 10 min, denaturation at 94 °C for 1 min, annealing tem- perature on the ship for five days. After suitable dilution of perature at 53 °C for 1 min, extension at 72 °C for 2 min, the cell cultures, the dilute was plated on YPD plates with final extension at 72 °C for 10 min. PCR was run for 32 0.05% chloramphenicol and the plates were incubated at cycles and PCR cycler was GeneAmp PCR System 2400 20-25 °C for five days. Different colonies from the plates made by Perkin-Elmer. PCR products were separated by were transferred to the YPD slants, respectively. agarose gel electrophoresis and recovered by using UNIQ- column DNA gel recovery kits (BIOASIA, Shanghai). The Lipase production. One loop of the cells of the yeast recovered PCR products were ligated into pGEM-T easy strains was transferred to 50 ml of YPD medium prepared vector and transformed into the competent cells of with distilled water in 250-ml flask and aerobically cultivat- Escherichia coli JM109. The transformants were selected on ed for 24 h. The cell culture (0.2 ml, OD = 20.0) was plates with ampicillin. The plasmids in the transformant 600nm transferred to 50 ml of the production medium which con- cells were extracted by using the methods as described by tained 3% (w/v) olive oil, 1% (w/v) ammonium sulphate, Sambrook et al. (1989). The D1/D2 26S rDNA fragments 0.2% (w/v) K HPO , 0.03% (w/v) MgSO ·7H O, 1.6% inserted on the vector were sequenced by Shanghai 2 4 4 2 (w/v) NaCl, 0.1% (w/v) yeast extract, pH 7.0 and grown by Sangon Company. shaking at 170 rpm and 25 °C for 4 days. The culture was centrifuged at 5000 rpm and 4 °C and the supernatant Phylogenetic analysis and identification of the yeasts. obtained was used as the crude lipase. For preparation of The sequences obtained above were aligned by using cell-bound lipase, the culture was washed three times by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST). For centrifugation at 5000 rpm and 4 °C and the cell pellets comparison with currently available sequences, several obtained were resuspended in 100 mM potassium phos- sequences were retrieved with over 98% similarity phate buffer (pH 7.0). belonging to different genera from NCBI (http://www.ncbi.nlm.nih.gov) and multiple alignment was Determination of lipase activity. The substrate emul- performed by using ClustalX 1.83 and phylogenetic trees sions were prepared by dropwise addition of 0.2 ml solution were constructed by PHYLIP 3.56. The routine identification A (40 mg of ρ-nitrophenyl-laurate was dissolved in 12 ml of the yeasts was performed by using the methods as isopropanol) into 3 ml solution B (0.4 g Triton X-100 and described by Kurtzman and Fell (2000). 0.1 g gum arabic were dissolved in 90 ml of 100 mM potas- sium phosphate buffer, pH 6.0-8.5) under intense vortex- Effects of pH and temperature on lipase activity. The ing. These emulsions were stable for 1 h at room temper- effects of pH on the enzyme activity were determined by ature. The crude lipase (0.1 ml) or the cell-bound lipase incubating the culture supernatant or the cell-bound lipase (0.1 ml) was added to 3.2 ml of the substrate emulsion and at different pH between 4.0 and 9.0 using the standard the mixture was incubated for 20 min in a shaking water assay conditions described above. The buffers used were bath at 35-40 °C. Then, the mixture was put into ice and 0.1 M citric-sodium dihydrogen phosphate buffer (pH 3.0), OD value at 410 nm in the mixture was read by using spec- 0.1 mM acetate buffer (pH 4.0-5.0), and 0.1 M phosphate trophotometer. The same mixture to which the same buffer (pH 6.0-9.0). The optimal temperature for activity of amount of the inactivated crude lipase or the inactivated the enzyme was determined at 30, 35, 40, 45, 50, 55, 60 cell-bound lipase (heated at 100 ºC for 10 min) was added and 65 °C in the same buffer as described above. before the reaction was used as the control. One unit (U) of enzyme activity was defined as the amount of enzyme Determination of hydrolytic activity of the lipases. ρ-nitrophenol per required for the liberation of 1.0 µM Five millilitres of 20 mM phosphate buffer (pH 6.0-8.5) and minute under the assay conditions. The specific lipase 4.0 ml of 50% oil emulsion were added to 100-ml flasks activity was units per mg of protein or per g of cell dry and the mixture was kept at 35-40 °C for 5 min. The crude weight. Protein concentration was measured by the method lipase (1 ml) or the cell-bound lipase (1 ml) was added to of Bradford, and bovine serum albumin served as standard the mixture in one of the flasks. Another flask without addi- (Bradford, 1976). Cell dry weight of the yeast culture was tion of the crude lipase or the cell-bound lipase was used measured according to the methods described by Chi et al. as the blank. The flasks were shaken at 35-40 °C in the (2001). water bath for 30 min. Ten millilitres of 95% ethanol solu- tion were added to the mixture immediately to cease the DNA extraction and PCR. The total genomic DNA of the reaction and 1.0 ml of the crude lipase or the cell-bound yeast strains was isolated and purified by using the meth- lipase was added to the blank. Liberated free fatty acids ods as described by Sambrook et al. (1989). Amplification were titrated with 0.05 M NaOH using phenolphthalein as and sequencing of 18S rDNA and ITS from the yeasts were indicator (Wu et al., 1996). One unit of hydrolytic activity performed according to the methods described by Chi et al. of the lipase was defined as the amount of enzyme which (2007). The common primers for amplification of D1/D2 catalyses the release of one µM of free fatty acids per min 26S rDNA in yeasts were used, the forward primer was NL- under the above conditions. The specific lipase activity was 1 (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and the reverse units per mg of protein or per g of cell dry weight. Ann. Microbiol., 57 (4), 495-501 (2007) 497 TABLE 1 - Sources of the lipase-producing marine yeasts from different marine environments grown in the medium with olive oil could produce lipase (data not shown). As Strains Sources shown in Table 1, the nine yeast strains were isolated from surface of Sargassum pallidum collected at Changdao YA01a Surface of Sargassum pallidum collected from Island, China, seawater at China South Sea, gut of seawater at Changdao Island, China Apostichopus japonicus from seawater in Srilanka, gut of N12c Seawater at China South Sea Pseudosciaena crocea collected at coastline in Qingdao, 3eA2 Gut of Apostichopus japonicus from seawater in salterns in Qingdao, deep seawater in South pole, surface Srilank of Laminaria japonica collected from seawater at YF12c Gut of Pseudosciaena crocea collected at the Changdao Island, China and gut of Nemipterus virgatus coastline in Qingdao collected at China East Sea, respectively. In recent years, JHSb Salterns in Qingdao we have found that diversity of marine yeasts is very rich. HN2.3 Salterns in Qingdao However, to our knowledge, the yeast from marine envi- N9a Deep seawater in South pole ronments is still an untouched bioresource for lipase pro- L10-2 Surface of Laminaria japonica collected from sea- duction (Chi et al., 2006). It is very interesting to observe water at Changdao Island, China from the results in Table 1 that lipase-producing yeasts Wl8 Gut of Nemipterus virgatus collected from sea- were distributed in many marine environments and many water at China East Sea species of lipase-producing yeasts occurred in the marine environments. Physiological and biochemical characterisation Based on the fermentation spectra and carbon source assimilation spectra of the yeasts isolated in this study and RESULTS AND DISCUSSION those of the type strains listed in The Yeast, A Taxonomic Study (Kurtzman and Fell, 2000), we found that strains Screening of the lipase-producing yeasts YA01a, N12c, 3eA2, YF12c, JHSb, HN2.3, N9a, L10-2 and Some of terrestrial yeasts have been confirmed to have w18 looked similar to Candida intermedia, Pichia guillier- the capacity to produce over 150 U/ml of lipase (Lotti et mondii, Candida parapsilosis, Lodderomyces elongisporus, al., 1998). Therefore, we want to know if such yeasts exist Candida quercitrusa, Aureobasidium pullulans, Yarrowia in marine environments. After over 400 yeast strains from lipolytica, Rhodotorula mucilaginosa and Candida rugosa, seawater, sediments, guts of the marine fish and marine respectively (Table 2). algae were screened, we found that nine yeast strains TABLE 2 - The fermentation and assimilation of different carbohydrates by the marine yeast strains Marine yeast strains YA01a N12c 3eA2 YF12c JHSb HN2.3 N9a L10-2 wl8 Fermentation Glucose + + + + ––––– Maltose ––––––––– Galactose + – + – + –––– Sucrose + ––––––– + Lactose ––––––––– Raffinose –––––––– + Melibiose ––––––––– Assimilation Glucose +++++++++ Maltose ++++++ – ++ Galactose +++++++++ Sucrose + +++++ – ++ Lactose + – W –––––– Raffinose + W W – + + – + + Melibiose – – – + + + – – – Amidulin W–W–W + – + + Trehalose + – ++++ – ++ Cellobiose + W W W + + – – – D–Arabinose W –––– + + + + Xylose ++++++ W ++ L–Arabinose W – ++++ – ++ +: positive; –: negative; W: weak. Fermentation and assimilation were performed at 25 °C in the media with 0.5% sugars at natural pH (Kutzman and Fell, 2000). 498 L. Wang et al. Phylogenetic analysis of partial sequences of the 18S rRNA genes, D1/D2 26S rDNA and ITS According to Kurtzman and Fell (2000), traditional and rou- tine identification methods which depend on phenotype, are usually leading to uncertain and inaccurate interpreta- tions of species interaction. Sequence analysis of phyloge- ny for microbial taxonomy, is a more accurate method for determining inter- and intra-specific relationships. Therefore, 18S rDNA partial sequences, D1/D2 26S rDNA sequences and ITS of the yeast strains were determined and aligned by using BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic trees were constructed by using PHYLIP software package ver- sion 3.56 (Felsenstein, 1995). Distance matrices were gen- erated by the DNADIST program, based on Kimura’s two- parameter model (Kimura, 1980). Neighbour-joining analy- FIG. 2 - Consensus tree of the isolates based on 9 D1/D2 26S sis of the data sets was carried out with the program rDNAs obtained in this study and 13 previously pub- Neighbour of the PHYLIP package. Cafeteria roenbergensis lished sequences obtained from GenBank. The outgroop (heterotrophic flagellates), Ricciocarpos natans and Gianda we used was Ricciocarpos natans. The numbers above intestinalis were used as out-groups during the construc- the branches are bootstrap, only values 96% are shown. tion of consensus trees of the isolates based on 18S rRNA gene sequences, D1/D2 26S rDNA sequences and ITS, respectively. The search for the similarity between 18S rDNA sequences, D1/D2 26S rDNA sequences and ITS of the isolates and those in the NCBI database shows that many phylogenetically related yeast species were similar to the yeast strains obtained in this study. Phylogenetic rela- tionships of 18S rDNA sequences, D1/D2 26S rDNA sequences and ITS of the yeast strains isolated in this study were shown in Figs. 1, 2 and 3 and their GenBank acces- sion numbers and the closest relatives are shown in Table 3. The topology of the phylogram in Figs.1, 2 and 3 con- firms that the strain YA01a was closely related Candida intermedia, whereas the strain N12C could be in close rela- tionship to Pichia guilliermondii. The strain 3eA2 was assigned to Candida parapsilosis. D1/D2 26S rDNA sequences and ITS of yeast strain YF12C were identical to those of Lodderomyces elongisporus, respectively. Strain FIG. 3 - Consensus tree of the isolates based on 8 ITS sequences obtained in this study and 13 previously published sequences obtained from GenBank. The outgroop we used was Gianda intestinalis. The numbers above the branches are bootstrap, only values > 99% are shown. JHSb was identified to be a strain of Candida quercitrusa. Strain HN2.3 was found to be a strain of Aureobasidium pullulans while strains N9a, L10-2 and w18 were similar to Yarrowia lipolytica, Rhodotorula mucilaginosa and Candida rugosa, respectively. The results were identical to those from fermentation spectra and carbon source assimilation spectra of the yeasts obtained in this study (Table 2). To date, only Candida rugosa, Candida tropicalis, Candida antarctica, Candida cylindracea, Candida parap- silosis, Candida deformans, Candida curvata, Candida vali- da, Yarrowia lipolytica, Rhodotorula glutinis, Rhodotorula pilimanae, Pichia bispora, Pichia mexicana, Pichia silvicola, FIG. 1 - Consensus tree of the isolates based on 6 18S rRNA Pichia xylosa, Pichia burtonii, Saccharomycopsis lipolytica, gene sequences obtained in this study and 9 previously Saccharomycopsis crataegenesis, Torulospora globosa and published sequences obtained from GenBank. The out- Trichosporon asteroides have been found to be able to pro- groop we used was Cafeteria roenbergensis. The num- duce lipase (Sharma et al., 2001; Vakhlu and Kour, 2006) bers above the branches are bootstrap, only values 98% among the terrestrial yeasts. Therefore, the yeast strains are shown. Ann. Microbiol., 57 (4), 495-501 (2007) 499 TABLE 3 - The accession numbers of ITS, 18S rDNA and D1/D2 26S rDNA from the nine lipase-producing marine yeasts Strains The closest relatives (% similarity) The accession numbers The accession numbers The accession numbers of 18S rRNA gene of ITS D1/D2 26S rRNA gene YA01a Candida intermedia (99% ) EF408189 DQ680837 EF362751 N12c Pichia guilliermondii (99% ) DQ438179 EF375704 EF375700 3eA2 Candida parapsilosis (over 99% ) EF208925 DQ681358 EF362748 YF12c Lodderomyces elongisporus (100% ) DQ515959 EF394941 EF394940 JHSb Candida quercitrusa (99% ) EF152413 DQ665264 EF375703 HN2.3 Aureobasidium pullulans (over 99% ) EF125668 DQ680685 EF375702 N9a Yarrowia lipolitica (100% ) EF190312 DQ683016 EF362750 L10-2 Rhodotorula mucilaginosa (over 99% ) EF218987 DQ681372 EF362749 wl8 Candida rugosa (over 99% ) EF371020 EF198009 EF375701 C. intermedia YA01a, L. elongisporus YF12c, P. guillier- produced by C. rugosa were 30 °C and pH 7.2, respective- mondii N12c, C. quercitrusa JHSb, R. mucilaginosa L10-2 ly (Lotti et al., 1998). This means that the optimal pH and and A. pullulans HN2.3 obtained in this study were the new temperature of the crude lipase from the yeasts obtained in producers of lipase. this study were in agreement with those from terrestrial yeasts. Effects of different temperature and pH on activity of the crude lipases Lipase activity and cellular location of lipases from The results in Table 4 show that the optimal pHs for the the yeasts crude lipases produced by the yeasts C. intermedia YA01a, It is very interesting to note from data in Table 5 that C. P. guilliermondii N12c, C. parapsilosis 3eA2 and L. elongis- intermedia YA01a, P. guilliermondii N12c, C. parapsilosis porus YF12c were 7.5 while the optimal pHs for the crude 3eA2, L. elongisporus YF12c, C. quercitrusa JHSb, C. lipases produced by the yeasts C. quercitrusa JHSb and C. rugosa wl8, Y. lipolytica N9a and R. mucilaginosa L10-2 rugosa wl8 were 7.0. It also can be seen from Table 4 that produced cell-bound lipase whereas A. pullulans HN2.3 the optimal pHs for the crude lipases from A. pullulans secreted lipase into the medium. Table 5 also shows that HN2.3, Y. lipolytica N9a and R. mucilaginosa L10-2 were the yeast strains C. intermedia YA01a and L. elongisporus 8.5, 8.0 and 6.0, respectively. It can be observed from the YF12c had higher cell-bound lipase activity than any other results in Table 4 that the optimal temperatures of the yeast strains tested in this study and A. pullulans HN2.3 crude lipases produced by the yeasts C. intermedia YA01a, had the highest extracellular lipase activity. C. parapsilosis 3eA2, L. elongisporus YF12c and Y. lipolyti- Our results (Table 5) show that majority of lipases from ca N9a were 40 ºC while the optimal temperatures of the the yeast strains used in this study were cell-bound and crude lipases produced by other yeast strains were 35 ºC, only A. pullulans HN2.3 secret a large amount of lipase into respectively. the medium. However, it has been confirmed that most of Usually, the optimal pH and temperature for lipase from lipases from terrestrial yeast are extracellular, but lipase terrestrial yeasts are between 5.0 and 8.0 and between 30 from terrestrial yeasts C. parapsilosis CBS 604 and some and 50 ºC (Vakhlu and Kour, 2006). For example, the lipase from Y. lipolytica are also cell-bound (Vakhlu and optimal pH and temperature for activity of the crude lipase Kour, 2006). TABLE 4 - The optimal temperature and pHs of the crude lipases TABLE 5 - The lipase activities produced by the marine yeast produced by the marine yeast strains strains and cellular location of lipases Strains Lipase activity Cellular Fermentation Strains Optimal temperature (ºC) Optimal pH -1 (U g ) location period (days) C. intermedia YA01a 40 7.5 C. intermedia YA01a 42.0 ± 1.2 Cell-bound 3 P. guilliermondii N12c 35 7.5 P. guilliermondii N12c 43.6 ± 1.8 Cell-bound 2 C. parapsilosis 3eA2 40 7.5 C. parapsilosis 3eA2 10.4 ± 0.12 Cell bound 3 L. elongisporus YF12c 40 7.5 L. elongisporus YF12c 16.6 ± 0.9 Cell bound 2 C. quercitrusa JHSb 35 7.0 C. quercitrusa JHSb 9.6 ± 0.3 Cell bound 4 A. pullulans HN2.3 35 8.5 A. pullulans HN2.3 8.2 ± 0.12 Extracellular 2 Y. lipolytica N9a 40 8.0 Y. lipolytica N9a 5.1 ± 0.73 Cell-bound 3 R. mucilaginosa L10-2 35 6.0 R. mucilaginosa L10-2 4.0 ± 0.23 Cell-bound 3 C. rugosa wl8 35 7.0 C. rugosa w18 26.9 ± 1.2 Cell-bound 2 Values are given as mean ± SD, n = 3. 500 L. Wang et al. TABLE 6 - Oil hydrolysis by the crude lipases Strains Olive oil Lard Peanut oil Soybean oil a a a a C. intermedia YA01a 200.0 ± 18.1 16.6 ± 2.4 160.0 ± 3.2 13.2 ± 0.5 a a a a P. guilliermondii N12c 21.0 ± 0.9 196.5 ± 2.7 119.5 ± 0.9 14.2 ± 1.1 a a a a C. parapsilosis 3eA2 94.1 ± 2.7 8.8 ± 0.5 10.1 ± 0.4 86.3 ± 3.5 a a a a L. elongisporus YF12c 74.4 ± 2.8 12.6 ± 0.7 93.0 ± 1.2 54.6 ± 0.5 a a a a C. quercitrusa JHSb 30.7 ± 3.2 4.3 ± 0.8 12.0 ± 0.1 8.3 ± 0.5 a a a a C. rugosa wl8 14.3 ± 0.6 6.8 ± 1.2 11.9 ± 1.5 9.1 ± 0.7 a a a a Y. lipolytica N9a 8.8 ± 0.4 3.3 ± 0.2 10.3 ± 0.8 13.5 ± 0.4 a a a a R. mucilaginosa L10-2 28.2 ± 1.7 7.4 ± 0.3 18.3 ± 0.9 16.7 ± 1.5 b b b b A. pullulans HN2.3 35.2 ± 1.3 33.2 ± 0.9 37.3 ± 1.9 29.6 ± 0.9 a b : units per g of cell dry weight; : units per mg of protein. Values are given as mean ± SD, n = 3. Hydrolytic activity of the crude lipases cell flocculation. The hydrolytic reaction catalysed by lipase generally takes In recent years, many results have also shown that place at the oil-water interface (Sharma et al., 2001). The lipase in the intestine of marine animals can help digest hydrolytic activity is the basic characteristic of lipase. It is lipid in their feed, inferring that lipase activity may be impli- required that lipase is non-specific, hydrolysing different cated in regulating the use of dietary components and pos- kinds of lipids from different sources when it is applied to sibly influencing the stages of development in marine ani- lipase biosensor, detergent industry and digestion of lipids mals (Fu et al., 2005). Therefore, marine yeasts with high in the food and medicine (Wu et al., 1996). Therefore, the lipase activity also can be used in mariculture. We also crude lipase hydrolytic activity by the marine yeasts think that lipase-producing marine yeasts are new biore- obtained in this study was assayed towards olive oil, souces and gene resources (Chi et al., 2006). peanut oil, soybean oil and lard. The results in Table 6 indi- cate that the crude lipase produced by the yeast strain Acknowledgements HN2.3 which could produce extracellular lipase had high This research was supported by Hi-Tech Research and hydrolytic activity towards all the oils, especially peanut oil Development Program of China (863), the grant No is while the cell-bound lipase from strain YA01a had high 2006AA09Z403. hydrolytic activity towards olive oil and peanut oil and cell- bound lipase from strain N12c had high hydrolytic activity REFFERENCES towards lard and peanut oil, suggesting that the extracel- lular lipase from strain HN2.3 and cell-bound lipases from Bradford M.M. (1976). A rapid and sensitive method for quanti- strains YA01a and N12c had highly potential application in tation of microgram quantities of protein utilizing the princi- digestion of lipids. However, cell-bound lipases from other ple of protein-dye binding. Anal. Biochem., 72: 248-253. yeast strains had very low hydrolytic activity towards the Chi Z.M., Liu J., Ji J.R., Meng Z. (2001). Trehalose accumulation oils tested in this study (Table 6). The results in Table 6 from starch by Saccharomycopsis fibuligera sdu. Enzyme. also show cell-bound lipases from strain YA01a had very Microb. Technol., 28: 240-245. low hydrolytic activity towards lard and soybean oil and Chi Z.M., Liu Z., Gao L., Gong F., Ma C., Wang X., Li H. (2006). cell-bound lipases from strain N12c had very low hydrolyt- Marine yeasts and their applications in mariculture. J. Ocean ic activity towards olive oil and soybean oil. Although lipase Univ. China 5: 251-256. from terrestrial yeast C. rugosa has been extensively used Chi Z., Ma C., Wang P., Li H. (2007). Optimization of medium in industrial and clinical purpose (Vakhlu and Kour, 2006), and cultivation conditions for alkaline protease production by lipase activity produced by the yeast C. rugosa wl8 the marine yeast Aureobasidium pullulans. Biores. Technol., 98: 534-538. obtained in this study was lower than that produced by C. Felsenstein J. (1995). PHYLIP (Phylogenetic Inference Package), intermedia YA01a and L. elongisporus YF12c tested in this Version 3.75. Distributed by author, Department of Genetics, study. University of Washinton, Seattle, WA. In China, a large amount of peanut oil and lard is dis- Fu X.Y., Xue C.H., Miao B.C., Li Z.T., Gao X., Yang W.G. (2005). charged from the restaurants and homes each day and Characterization of protease from the digestive tract of sea causes heavy pollution in fresh water and seawater. cucumber (Stichopus japonicus): high alkaline protease Therefore, the lipase with high hydrolytic activity towards activity. Aquaculture, 246: 321-329. peanut oil and lard may have highly potential applications Hasan F., Shah A.A., Hameed A. (2006). Industrial applications in degradation of oil in fresh water and seawater and reuse of microbial lipases. Enzyme. Microb. Technol., 39: 235-251. of the wasted peanut oil. It should be stressed that the cell- Kimura M. (1980). A simple method for estimating evolutionary bound lipase from the marine yeasts had many advantages rate of base substitutions through comparative studies on over the extracellular lipase when it is applied to industries nucleotide sequences. J. Mol. E., 2: 87-90. because it is very easy to collect and concentrate the cell- Kurtzman C.P., Fell J.W. (2000). The Yeasts. A Taxonomic Study, th bound lipase by centrifugation of yeast culture and yeast 4 revised and enlarged edn., Elsevier, Amsterdam, Ann. Microbiol., 57 (4), 495-501 (2007) 501 Lausanne, New York, Oxford, Shannon, Singapore, Tokyo. Cold Spring Harbor Laboratory Press, Beijing, pp. 367-370. Lotti M., Monticelli S., Montesinos J.L., Brocca S., Valero F., Sugita T., Takashima M., Kodama M., Tsuboi R., Nishikawa A. Lafuente J. (1998). Physiological control on the expression (2003). Description of a new yeast species, Malassezia and secretion of Candida rugosa lipase. Chem. Phys. Lipids, japonica, and its detection in patients with atopic dermatitis 93: 143-148. and healthy subjects. J. Clin. Microbiol., 40: 4695-4699. Sharma R., Chistib Y., Banerjee U.C. (2001). Production, purifi- Vakhlu J., Kour A. (2006). Yeast lipases: enzyme purification, cation, characterization and applications of lipases. biochemical properties and gene cloning. E. J. Biotechnol., 9: Biotechnol. Adv., 19: 627-662. 1-17. Sambrook J., Fritsch E.F., Maniatis T. (1989). Molecular Cloning: Wu X.Y., Jaaskelainen S., Linko Y. (1996). An investigation of nd A Laboratory Manual, 2 edn. (Chinese translating edn.), crude lipase for hydrolysis, esterization and transesterifica- http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes

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Springer Journals
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Copyright © 2007 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/BF03175345
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Abstract

Annals of Microbiology, 57 (4) 495-501 (2007) Diversity of lipase-producing yeasts from marine environments and oil hydrolysis by their crude enzymes Lin WANG, Zhenming CHI*, Xianghong WANG, Zhiqiang LIU, Jing LI Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China Received 15 May 2007 / Accepted 27 September 2007 Abstract - Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish and marine algae were obtained. After lipase activity of the yeast cultures was estimated, we found that nine yeast strains obtained in this study grown in the medium with olive oil could produce lipase. The results of routine identification and molecular methods show that they belonged to Candida intermedia YA01a, Pichia guilliermondii N12c, Candida parapsilosis 3eA2, Lodderomyces elongisporus YF12c, Candida quercitrusa JHSb, Candia rugosa wl8, Yarrowia lipolytica N9a, Rhodotorula mucilaginosa L10-2 and Aureobasidium pullulans HN2.3, respectively. The optimal pHs and temperatures of lipases produced by them were between 6.0 and 8.5 and between 35 and 40 ºC, respectively. Majority of lipases from the yeast strains were cell-bound and only lipase from A. pullulans HN2.3 was extracellular. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry. Key words: lipase, marine-derived yeasts, diversity, marine environments, lipid hydrolysis. INTRODUCTION been purified and characterised and the genes encoding lipase in Candida, Geotrichum, Trichosporon and Y. lipolyt- Lipases are a class of hydrolyases that are primarily ica have been cloned and overexpressed (Vakhlu and Kour, responsible for the hydrolysis and the synthesis of esters 2006; Sharma et al., 2001). It has been found that most of formed from glycerol and long-chain fatty acids in vitro and lipases are serine hydrolyases according to their biochemi- in vivo. They catalyse a wide range of reactions, including cal properties (Vakhlu and Kour, 2006). Although lipases hydrolysis, inter-esterification, alcoholysis, acidolysis, from Candida rugosa and Candida antarctica have been esterification and aminolysis. Therefore, lipases, especially extensively used in flavour industry, synthesis of lipophillic microbial lipases have many industrial applications in the antioxidants, detergent industry, lipid hydrolysis, biosen- detergent, food, flavour industry, biocatalytic resolution of sors and clinical purpose, very few studies exist on the pharmaceuticals, esters and amino acid derivatives, mak- lipase produced by the yeasts obtained from marine envi- ing of fine chemicals, agrochemicals, use as biosensor, ronments (Chi et al., 2006). bioremediation and cosmetics and perfumery (Hasan et al., After we screened over 400 yeast strains from different 2006). Following proteases and carbohydrases, lipases are marine environments, we found that some yeast strains considered to be the third largest group based on total from different marine environments could produce lipase. sales volume. The commercial use of lipases is a billion-dol- The main purpose of the present study was to analyse lar business that comprises a wide variety of different appli- diversity of lipase-producing yeasts from different marine cations. Usually, extracellular lipase is produced by sub- environments. We also carried out the hydrolysis of various merged liquid fermentation, solid state fermentation and oils by the crude lipases or cell-bound lipases produced by immobilised cell culture (Sharma et al., 2001). Numerous the lipase-producing yeasts. To our knowledge, this is the species of bacteria, yeasts and molds were found to pro- first report on the lipase-producing yeasts derived from the duce lipases. Among the terrestrial yeasts, several Candida marine environments. spp., Yarrowia lipolytica, several Rhodotorula spp., some Pichia spp., Saccharomycopsis crataegensis, Torulospora globosa and Trichosporon asteroides have been found to be MATERIALS AND METHODS able to produce lipase (Vakhlu and Kour, 2006; Sharma et al., 2001). Because most of yeast strains are considered as Sampling. Different samples of seawater and sediments non-pathogenic, the processes for lipase production based in China South Sea, China East Sea, Indian Ocean and the on yeasts have been classified as GRAS (generally regard- Pacific Ocean were collected during the Antarctic explo- ed as safe). Extracellular lipases from several yeasts have ration in 2004 and hypersaline sea water, sediments of the salterns, different species of marine animals and algae along the coast of Qingdao, China were also col- * Corresponding author. Phone and Fax: 0086-532-82032266; lected. E-mail: zhenming@sdu.edu.cn 496 L. Wang et al. Isolation of marine yeasts. Two millilitres of the seawa- primer was NL-4 (5’-GGTCCGTGTTTCAAGACGG-3’) (Sugita ter or 2 g of the sediments or 2 ml of homogenised guts of et al., 2003). The reaction system (25 µl) was composed of marine animals or homogenised marine algae were sus- 10x buffer 2.5 µl, dNTP 0.8 µM, MgCl 1.5 mM, NL-1 0.5 pended in 20 ml of YPD medium containing 2% (w/v) glu- µM, NL-4 0.5 µM, Taq DNA polymerase 1.25 U, template cose, 2% (w/v) polypeptone and 1% (w/v) yeast extract DNA 1.0 µl and H O 16.6 µl. The conditions for the PCR and supplemented with 0.05% (w/v) chloramphenicol amplification were as follows: initial denaturation at 94 °C immediately after sampling and cultivated at natural tem- for 10 min, denaturation at 94 °C for 1 min, annealing tem- perature on the ship for five days. After suitable dilution of perature at 53 °C for 1 min, extension at 72 °C for 2 min, the cell cultures, the dilute was plated on YPD plates with final extension at 72 °C for 10 min. PCR was run for 32 0.05% chloramphenicol and the plates were incubated at cycles and PCR cycler was GeneAmp PCR System 2400 20-25 °C for five days. Different colonies from the plates made by Perkin-Elmer. PCR products were separated by were transferred to the YPD slants, respectively. agarose gel electrophoresis and recovered by using UNIQ- column DNA gel recovery kits (BIOASIA, Shanghai). The Lipase production. One loop of the cells of the yeast recovered PCR products were ligated into pGEM-T easy strains was transferred to 50 ml of YPD medium prepared vector and transformed into the competent cells of with distilled water in 250-ml flask and aerobically cultivat- Escherichia coli JM109. The transformants were selected on ed for 24 h. The cell culture (0.2 ml, OD = 20.0) was plates with ampicillin. The plasmids in the transformant 600nm transferred to 50 ml of the production medium which con- cells were extracted by using the methods as described by tained 3% (w/v) olive oil, 1% (w/v) ammonium sulphate, Sambrook et al. (1989). The D1/D2 26S rDNA fragments 0.2% (w/v) K HPO , 0.03% (w/v) MgSO ·7H O, 1.6% inserted on the vector were sequenced by Shanghai 2 4 4 2 (w/v) NaCl, 0.1% (w/v) yeast extract, pH 7.0 and grown by Sangon Company. shaking at 170 rpm and 25 °C for 4 days. The culture was centrifuged at 5000 rpm and 4 °C and the supernatant Phylogenetic analysis and identification of the yeasts. obtained was used as the crude lipase. For preparation of The sequences obtained above were aligned by using cell-bound lipase, the culture was washed three times by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST). For centrifugation at 5000 rpm and 4 °C and the cell pellets comparison with currently available sequences, several obtained were resuspended in 100 mM potassium phos- sequences were retrieved with over 98% similarity phate buffer (pH 7.0). belonging to different genera from NCBI (http://www.ncbi.nlm.nih.gov) and multiple alignment was Determination of lipase activity. The substrate emul- performed by using ClustalX 1.83 and phylogenetic trees sions were prepared by dropwise addition of 0.2 ml solution were constructed by PHYLIP 3.56. The routine identification A (40 mg of ρ-nitrophenyl-laurate was dissolved in 12 ml of the yeasts was performed by using the methods as isopropanol) into 3 ml solution B (0.4 g Triton X-100 and described by Kurtzman and Fell (2000). 0.1 g gum arabic were dissolved in 90 ml of 100 mM potas- sium phosphate buffer, pH 6.0-8.5) under intense vortex- Effects of pH and temperature on lipase activity. The ing. These emulsions were stable for 1 h at room temper- effects of pH on the enzyme activity were determined by ature. The crude lipase (0.1 ml) or the cell-bound lipase incubating the culture supernatant or the cell-bound lipase (0.1 ml) was added to 3.2 ml of the substrate emulsion and at different pH between 4.0 and 9.0 using the standard the mixture was incubated for 20 min in a shaking water assay conditions described above. The buffers used were bath at 35-40 °C. Then, the mixture was put into ice and 0.1 M citric-sodium dihydrogen phosphate buffer (pH 3.0), OD value at 410 nm in the mixture was read by using spec- 0.1 mM acetate buffer (pH 4.0-5.0), and 0.1 M phosphate trophotometer. The same mixture to which the same buffer (pH 6.0-9.0). The optimal temperature for activity of amount of the inactivated crude lipase or the inactivated the enzyme was determined at 30, 35, 40, 45, 50, 55, 60 cell-bound lipase (heated at 100 ºC for 10 min) was added and 65 °C in the same buffer as described above. before the reaction was used as the control. One unit (U) of enzyme activity was defined as the amount of enzyme Determination of hydrolytic activity of the lipases. ρ-nitrophenol per required for the liberation of 1.0 µM Five millilitres of 20 mM phosphate buffer (pH 6.0-8.5) and minute under the assay conditions. The specific lipase 4.0 ml of 50% oil emulsion were added to 100-ml flasks activity was units per mg of protein or per g of cell dry and the mixture was kept at 35-40 °C for 5 min. The crude weight. Protein concentration was measured by the method lipase (1 ml) or the cell-bound lipase (1 ml) was added to of Bradford, and bovine serum albumin served as standard the mixture in one of the flasks. Another flask without addi- (Bradford, 1976). Cell dry weight of the yeast culture was tion of the crude lipase or the cell-bound lipase was used measured according to the methods described by Chi et al. as the blank. The flasks were shaken at 35-40 °C in the (2001). water bath for 30 min. Ten millilitres of 95% ethanol solu- tion were added to the mixture immediately to cease the DNA extraction and PCR. The total genomic DNA of the reaction and 1.0 ml of the crude lipase or the cell-bound yeast strains was isolated and purified by using the meth- lipase was added to the blank. Liberated free fatty acids ods as described by Sambrook et al. (1989). Amplification were titrated with 0.05 M NaOH using phenolphthalein as and sequencing of 18S rDNA and ITS from the yeasts were indicator (Wu et al., 1996). One unit of hydrolytic activity performed according to the methods described by Chi et al. of the lipase was defined as the amount of enzyme which (2007). The common primers for amplification of D1/D2 catalyses the release of one µM of free fatty acids per min 26S rDNA in yeasts were used, the forward primer was NL- under the above conditions. The specific lipase activity was 1 (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and the reverse units per mg of protein or per g of cell dry weight. Ann. Microbiol., 57 (4), 495-501 (2007) 497 TABLE 1 - Sources of the lipase-producing marine yeasts from different marine environments grown in the medium with olive oil could produce lipase (data not shown). As Strains Sources shown in Table 1, the nine yeast strains were isolated from surface of Sargassum pallidum collected at Changdao YA01a Surface of Sargassum pallidum collected from Island, China, seawater at China South Sea, gut of seawater at Changdao Island, China Apostichopus japonicus from seawater in Srilanka, gut of N12c Seawater at China South Sea Pseudosciaena crocea collected at coastline in Qingdao, 3eA2 Gut of Apostichopus japonicus from seawater in salterns in Qingdao, deep seawater in South pole, surface Srilank of Laminaria japonica collected from seawater at YF12c Gut of Pseudosciaena crocea collected at the Changdao Island, China and gut of Nemipterus virgatus coastline in Qingdao collected at China East Sea, respectively. In recent years, JHSb Salterns in Qingdao we have found that diversity of marine yeasts is very rich. HN2.3 Salterns in Qingdao However, to our knowledge, the yeast from marine envi- N9a Deep seawater in South pole ronments is still an untouched bioresource for lipase pro- L10-2 Surface of Laminaria japonica collected from sea- duction (Chi et al., 2006). It is very interesting to observe water at Changdao Island, China from the results in Table 1 that lipase-producing yeasts Wl8 Gut of Nemipterus virgatus collected from sea- were distributed in many marine environments and many water at China East Sea species of lipase-producing yeasts occurred in the marine environments. Physiological and biochemical characterisation Based on the fermentation spectra and carbon source assimilation spectra of the yeasts isolated in this study and RESULTS AND DISCUSSION those of the type strains listed in The Yeast, A Taxonomic Study (Kurtzman and Fell, 2000), we found that strains Screening of the lipase-producing yeasts YA01a, N12c, 3eA2, YF12c, JHSb, HN2.3, N9a, L10-2 and Some of terrestrial yeasts have been confirmed to have w18 looked similar to Candida intermedia, Pichia guillier- the capacity to produce over 150 U/ml of lipase (Lotti et mondii, Candida parapsilosis, Lodderomyces elongisporus, al., 1998). Therefore, we want to know if such yeasts exist Candida quercitrusa, Aureobasidium pullulans, Yarrowia in marine environments. After over 400 yeast strains from lipolytica, Rhodotorula mucilaginosa and Candida rugosa, seawater, sediments, guts of the marine fish and marine respectively (Table 2). algae were screened, we found that nine yeast strains TABLE 2 - The fermentation and assimilation of different carbohydrates by the marine yeast strains Marine yeast strains YA01a N12c 3eA2 YF12c JHSb HN2.3 N9a L10-2 wl8 Fermentation Glucose + + + + ––––– Maltose ––––––––– Galactose + – + – + –––– Sucrose + ––––––– + Lactose ––––––––– Raffinose –––––––– + Melibiose ––––––––– Assimilation Glucose +++++++++ Maltose ++++++ – ++ Galactose +++++++++ Sucrose + +++++ – ++ Lactose + – W –––––– Raffinose + W W – + + – + + Melibiose – – – + + + – – – Amidulin W–W–W + – + + Trehalose + – ++++ – ++ Cellobiose + W W W + + – – – D–Arabinose W –––– + + + + Xylose ++++++ W ++ L–Arabinose W – ++++ – ++ +: positive; –: negative; W: weak. Fermentation and assimilation were performed at 25 °C in the media with 0.5% sugars at natural pH (Kutzman and Fell, 2000). 498 L. Wang et al. Phylogenetic analysis of partial sequences of the 18S rRNA genes, D1/D2 26S rDNA and ITS According to Kurtzman and Fell (2000), traditional and rou- tine identification methods which depend on phenotype, are usually leading to uncertain and inaccurate interpreta- tions of species interaction. Sequence analysis of phyloge- ny for microbial taxonomy, is a more accurate method for determining inter- and intra-specific relationships. Therefore, 18S rDNA partial sequences, D1/D2 26S rDNA sequences and ITS of the yeast strains were determined and aligned by using BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic trees were constructed by using PHYLIP software package ver- sion 3.56 (Felsenstein, 1995). Distance matrices were gen- erated by the DNADIST program, based on Kimura’s two- parameter model (Kimura, 1980). Neighbour-joining analy- FIG. 2 - Consensus tree of the isolates based on 9 D1/D2 26S sis of the data sets was carried out with the program rDNAs obtained in this study and 13 previously pub- Neighbour of the PHYLIP package. Cafeteria roenbergensis lished sequences obtained from GenBank. The outgroop (heterotrophic flagellates), Ricciocarpos natans and Gianda we used was Ricciocarpos natans. The numbers above intestinalis were used as out-groups during the construc- the branches are bootstrap, only values 96% are shown. tion of consensus trees of the isolates based on 18S rRNA gene sequences, D1/D2 26S rDNA sequences and ITS, respectively. The search for the similarity between 18S rDNA sequences, D1/D2 26S rDNA sequences and ITS of the isolates and those in the NCBI database shows that many phylogenetically related yeast species were similar to the yeast strains obtained in this study. Phylogenetic rela- tionships of 18S rDNA sequences, D1/D2 26S rDNA sequences and ITS of the yeast strains isolated in this study were shown in Figs. 1, 2 and 3 and their GenBank acces- sion numbers and the closest relatives are shown in Table 3. The topology of the phylogram in Figs.1, 2 and 3 con- firms that the strain YA01a was closely related Candida intermedia, whereas the strain N12C could be in close rela- tionship to Pichia guilliermondii. The strain 3eA2 was assigned to Candida parapsilosis. D1/D2 26S rDNA sequences and ITS of yeast strain YF12C were identical to those of Lodderomyces elongisporus, respectively. Strain FIG. 3 - Consensus tree of the isolates based on 8 ITS sequences obtained in this study and 13 previously published sequences obtained from GenBank. The outgroop we used was Gianda intestinalis. The numbers above the branches are bootstrap, only values > 99% are shown. JHSb was identified to be a strain of Candida quercitrusa. Strain HN2.3 was found to be a strain of Aureobasidium pullulans while strains N9a, L10-2 and w18 were similar to Yarrowia lipolytica, Rhodotorula mucilaginosa and Candida rugosa, respectively. The results were identical to those from fermentation spectra and carbon source assimilation spectra of the yeasts obtained in this study (Table 2). To date, only Candida rugosa, Candida tropicalis, Candida antarctica, Candida cylindracea, Candida parap- silosis, Candida deformans, Candida curvata, Candida vali- da, Yarrowia lipolytica, Rhodotorula glutinis, Rhodotorula pilimanae, Pichia bispora, Pichia mexicana, Pichia silvicola, FIG. 1 - Consensus tree of the isolates based on 6 18S rRNA Pichia xylosa, Pichia burtonii, Saccharomycopsis lipolytica, gene sequences obtained in this study and 9 previously Saccharomycopsis crataegenesis, Torulospora globosa and published sequences obtained from GenBank. The out- Trichosporon asteroides have been found to be able to pro- groop we used was Cafeteria roenbergensis. The num- duce lipase (Sharma et al., 2001; Vakhlu and Kour, 2006) bers above the branches are bootstrap, only values 98% among the terrestrial yeasts. Therefore, the yeast strains are shown. Ann. Microbiol., 57 (4), 495-501 (2007) 499 TABLE 3 - The accession numbers of ITS, 18S rDNA and D1/D2 26S rDNA from the nine lipase-producing marine yeasts Strains The closest relatives (% similarity) The accession numbers The accession numbers The accession numbers of 18S rRNA gene of ITS D1/D2 26S rRNA gene YA01a Candida intermedia (99% ) EF408189 DQ680837 EF362751 N12c Pichia guilliermondii (99% ) DQ438179 EF375704 EF375700 3eA2 Candida parapsilosis (over 99% ) EF208925 DQ681358 EF362748 YF12c Lodderomyces elongisporus (100% ) DQ515959 EF394941 EF394940 JHSb Candida quercitrusa (99% ) EF152413 DQ665264 EF375703 HN2.3 Aureobasidium pullulans (over 99% ) EF125668 DQ680685 EF375702 N9a Yarrowia lipolitica (100% ) EF190312 DQ683016 EF362750 L10-2 Rhodotorula mucilaginosa (over 99% ) EF218987 DQ681372 EF362749 wl8 Candida rugosa (over 99% ) EF371020 EF198009 EF375701 C. intermedia YA01a, L. elongisporus YF12c, P. guillier- produced by C. rugosa were 30 °C and pH 7.2, respective- mondii N12c, C. quercitrusa JHSb, R. mucilaginosa L10-2 ly (Lotti et al., 1998). This means that the optimal pH and and A. pullulans HN2.3 obtained in this study were the new temperature of the crude lipase from the yeasts obtained in producers of lipase. this study were in agreement with those from terrestrial yeasts. Effects of different temperature and pH on activity of the crude lipases Lipase activity and cellular location of lipases from The results in Table 4 show that the optimal pHs for the the yeasts crude lipases produced by the yeasts C. intermedia YA01a, It is very interesting to note from data in Table 5 that C. P. guilliermondii N12c, C. parapsilosis 3eA2 and L. elongis- intermedia YA01a, P. guilliermondii N12c, C. parapsilosis porus YF12c were 7.5 while the optimal pHs for the crude 3eA2, L. elongisporus YF12c, C. quercitrusa JHSb, C. lipases produced by the yeasts C. quercitrusa JHSb and C. rugosa wl8, Y. lipolytica N9a and R. mucilaginosa L10-2 rugosa wl8 were 7.0. It also can be seen from Table 4 that produced cell-bound lipase whereas A. pullulans HN2.3 the optimal pHs for the crude lipases from A. pullulans secreted lipase into the medium. Table 5 also shows that HN2.3, Y. lipolytica N9a and R. mucilaginosa L10-2 were the yeast strains C. intermedia YA01a and L. elongisporus 8.5, 8.0 and 6.0, respectively. It can be observed from the YF12c had higher cell-bound lipase activity than any other results in Table 4 that the optimal temperatures of the yeast strains tested in this study and A. pullulans HN2.3 crude lipases produced by the yeasts C. intermedia YA01a, had the highest extracellular lipase activity. C. parapsilosis 3eA2, L. elongisporus YF12c and Y. lipolyti- Our results (Table 5) show that majority of lipases from ca N9a were 40 ºC while the optimal temperatures of the the yeast strains used in this study were cell-bound and crude lipases produced by other yeast strains were 35 ºC, only A. pullulans HN2.3 secret a large amount of lipase into respectively. the medium. However, it has been confirmed that most of Usually, the optimal pH and temperature for lipase from lipases from terrestrial yeast are extracellular, but lipase terrestrial yeasts are between 5.0 and 8.0 and between 30 from terrestrial yeasts C. parapsilosis CBS 604 and some and 50 ºC (Vakhlu and Kour, 2006). For example, the lipase from Y. lipolytica are also cell-bound (Vakhlu and optimal pH and temperature for activity of the crude lipase Kour, 2006). TABLE 4 - The optimal temperature and pHs of the crude lipases TABLE 5 - The lipase activities produced by the marine yeast produced by the marine yeast strains strains and cellular location of lipases Strains Lipase activity Cellular Fermentation Strains Optimal temperature (ºC) Optimal pH -1 (U g ) location period (days) C. intermedia YA01a 40 7.5 C. intermedia YA01a 42.0 ± 1.2 Cell-bound 3 P. guilliermondii N12c 35 7.5 P. guilliermondii N12c 43.6 ± 1.8 Cell-bound 2 C. parapsilosis 3eA2 40 7.5 C. parapsilosis 3eA2 10.4 ± 0.12 Cell bound 3 L. elongisporus YF12c 40 7.5 L. elongisporus YF12c 16.6 ± 0.9 Cell bound 2 C. quercitrusa JHSb 35 7.0 C. quercitrusa JHSb 9.6 ± 0.3 Cell bound 4 A. pullulans HN2.3 35 8.5 A. pullulans HN2.3 8.2 ± 0.12 Extracellular 2 Y. lipolytica N9a 40 8.0 Y. lipolytica N9a 5.1 ± 0.73 Cell-bound 3 R. mucilaginosa L10-2 35 6.0 R. mucilaginosa L10-2 4.0 ± 0.23 Cell-bound 3 C. rugosa wl8 35 7.0 C. rugosa w18 26.9 ± 1.2 Cell-bound 2 Values are given as mean ± SD, n = 3. 500 L. Wang et al. TABLE 6 - Oil hydrolysis by the crude lipases Strains Olive oil Lard Peanut oil Soybean oil a a a a C. intermedia YA01a 200.0 ± 18.1 16.6 ± 2.4 160.0 ± 3.2 13.2 ± 0.5 a a a a P. guilliermondii N12c 21.0 ± 0.9 196.5 ± 2.7 119.5 ± 0.9 14.2 ± 1.1 a a a a C. parapsilosis 3eA2 94.1 ± 2.7 8.8 ± 0.5 10.1 ± 0.4 86.3 ± 3.5 a a a a L. elongisporus YF12c 74.4 ± 2.8 12.6 ± 0.7 93.0 ± 1.2 54.6 ± 0.5 a a a a C. quercitrusa JHSb 30.7 ± 3.2 4.3 ± 0.8 12.0 ± 0.1 8.3 ± 0.5 a a a a C. rugosa wl8 14.3 ± 0.6 6.8 ± 1.2 11.9 ± 1.5 9.1 ± 0.7 a a a a Y. lipolytica N9a 8.8 ± 0.4 3.3 ± 0.2 10.3 ± 0.8 13.5 ± 0.4 a a a a R. mucilaginosa L10-2 28.2 ± 1.7 7.4 ± 0.3 18.3 ± 0.9 16.7 ± 1.5 b b b b A. pullulans HN2.3 35.2 ± 1.3 33.2 ± 0.9 37.3 ± 1.9 29.6 ± 0.9 a b : units per g of cell dry weight; : units per mg of protein. Values are given as mean ± SD, n = 3. Hydrolytic activity of the crude lipases cell flocculation. The hydrolytic reaction catalysed by lipase generally takes In recent years, many results have also shown that place at the oil-water interface (Sharma et al., 2001). The lipase in the intestine of marine animals can help digest hydrolytic activity is the basic characteristic of lipase. It is lipid in their feed, inferring that lipase activity may be impli- required that lipase is non-specific, hydrolysing different cated in regulating the use of dietary components and pos- kinds of lipids from different sources when it is applied to sibly influencing the stages of development in marine ani- lipase biosensor, detergent industry and digestion of lipids mals (Fu et al., 2005). Therefore, marine yeasts with high in the food and medicine (Wu et al., 1996). Therefore, the lipase activity also can be used in mariculture. We also crude lipase hydrolytic activity by the marine yeasts think that lipase-producing marine yeasts are new biore- obtained in this study was assayed towards olive oil, souces and gene resources (Chi et al., 2006). peanut oil, soybean oil and lard. The results in Table 6 indi- cate that the crude lipase produced by the yeast strain Acknowledgements HN2.3 which could produce extracellular lipase had high This research was supported by Hi-Tech Research and hydrolytic activity towards all the oils, especially peanut oil Development Program of China (863), the grant No is while the cell-bound lipase from strain YA01a had high 2006AA09Z403. hydrolytic activity towards olive oil and peanut oil and cell- bound lipase from strain N12c had high hydrolytic activity REFFERENCES towards lard and peanut oil, suggesting that the extracel- lular lipase from strain HN2.3 and cell-bound lipases from Bradford M.M. (1976). A rapid and sensitive method for quanti- strains YA01a and N12c had highly potential application in tation of microgram quantities of protein utilizing the princi- digestion of lipids. However, cell-bound lipases from other ple of protein-dye binding. Anal. Biochem., 72: 248-253. yeast strains had very low hydrolytic activity towards the Chi Z.M., Liu J., Ji J.R., Meng Z. (2001). Trehalose accumulation oils tested in this study (Table 6). The results in Table 6 from starch by Saccharomycopsis fibuligera sdu. Enzyme. also show cell-bound lipases from strain YA01a had very Microb. Technol., 28: 240-245. low hydrolytic activity towards lard and soybean oil and Chi Z.M., Liu Z., Gao L., Gong F., Ma C., Wang X., Li H. (2006). cell-bound lipases from strain N12c had very low hydrolyt- Marine yeasts and their applications in mariculture. J. Ocean ic activity towards olive oil and soybean oil. Although lipase Univ. China 5: 251-256. from terrestrial yeast C. rugosa has been extensively used Chi Z., Ma C., Wang P., Li H. (2007). Optimization of medium in industrial and clinical purpose (Vakhlu and Kour, 2006), and cultivation conditions for alkaline protease production by lipase activity produced by the yeast C. rugosa wl8 the marine yeast Aureobasidium pullulans. Biores. Technol., 98: 534-538. obtained in this study was lower than that produced by C. Felsenstein J. (1995). PHYLIP (Phylogenetic Inference Package), intermedia YA01a and L. elongisporus YF12c tested in this Version 3.75. Distributed by author, Department of Genetics, study. University of Washinton, Seattle, WA. In China, a large amount of peanut oil and lard is dis- Fu X.Y., Xue C.H., Miao B.C., Li Z.T., Gao X., Yang W.G. (2005). charged from the restaurants and homes each day and Characterization of protease from the digestive tract of sea causes heavy pollution in fresh water and seawater. cucumber (Stichopus japonicus): high alkaline protease Therefore, the lipase with high hydrolytic activity towards activity. Aquaculture, 246: 321-329. peanut oil and lard may have highly potential applications Hasan F., Shah A.A., Hameed A. (2006). Industrial applications in degradation of oil in fresh water and seawater and reuse of microbial lipases. Enzyme. Microb. Technol., 39: 235-251. of the wasted peanut oil. It should be stressed that the cell- Kimura M. (1980). A simple method for estimating evolutionary bound lipase from the marine yeasts had many advantages rate of base substitutions through comparative studies on over the extracellular lipase when it is applied to industries nucleotide sequences. J. Mol. E., 2: 87-90. because it is very easy to collect and concentrate the cell- Kurtzman C.P., Fell J.W. (2000). The Yeasts. A Taxonomic Study, th bound lipase by centrifugation of yeast culture and yeast 4 revised and enlarged edn., Elsevier, Amsterdam, Ann. Microbiol., 57 (4), 495-501 (2007) 501 Lausanne, New York, Oxford, Shannon, Singapore, Tokyo. Cold Spring Harbor Laboratory Press, Beijing, pp. 367-370. Lotti M., Monticelli S., Montesinos J.L., Brocca S., Valero F., Sugita T., Takashima M., Kodama M., Tsuboi R., Nishikawa A. Lafuente J. (1998). Physiological control on the expression (2003). Description of a new yeast species, Malassezia and secretion of Candida rugosa lipase. Chem. Phys. Lipids, japonica, and its detection in patients with atopic dermatitis 93: 143-148. and healthy subjects. J. Clin. Microbiol., 40: 4695-4699. Sharma R., Chistib Y., Banerjee U.C. (2001). Production, purifi- Vakhlu J., Kour A. (2006). Yeast lipases: enzyme purification, cation, characterization and applications of lipases. biochemical properties and gene cloning. E. J. Biotechnol., 9: Biotechnol. Adv., 19: 627-662. 1-17. Sambrook J., Fritsch E.F., Maniatis T. (1989). Molecular Cloning: Wu X.Y., Jaaskelainen S., Linko Y. (1996). An investigation of nd A Laboratory Manual, 2 edn. (Chinese translating edn.), crude lipase for hydrolysis, esterization and transesterifica-

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Published: Nov 21, 2009

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