World Journal

of Microbiology

& Biotechnology

10. 367-373

Molecular forms of lipases and their localization in the fungus Rhizopus microsporus by immuno-electron microscopy J.K. Diyorov, K.A. Lusta,* A.B. Tsiomenko and I.S. Kulaev Several lipases differing in their molecular masses (24, 32, 43, 66 and 98 kDa and 28,40,45 and 69 kDa) were found in Rhizopus microsporus UzLT-4B and UzLT-5C, respectively. The lipases in each strain were immunologically related. Strain UzLT-5C grown on a medium with lipid substrate secreted lipases of 32,66 and 98 kDa whereas strain UzLT-4B produced lipases of 45 and 69 kDa on the same medium. Immuno-electron microscopy indicated that the intracellular lipases were in peripheral zones of the hyphae, primarily in the periplasm and adjacent vesicles. Immobilization in Ca-alginate gel revealed unusual structures in the cell wall. These structures accumulated lipases and, apparently, exported the enzymes out of the cell. Key words: Filamentous

fungi, immunocytochemical

localization,

Filamentous fungi produce many enzymes, including lipases (Rivera-Munoz et al. 1991). As a rule, such fungi contain multiple forms of lipase (Huge-Jensen et al. 1987) and these are mostly secreted. The genetic and biochemical properties of lipases have been investigated extensively but the mechanism of their secretion is still unclear. Many studies have shown that macromolecules with M, of 15 to 20 kDa (Trevithick & Metzenberg 1966; Money 1990) can pass through the cell wall pores. However, fungi secrete enzymes of 210 kDa (Meachum et al. 1971), far larger than this free penetration limit. In filamentous fungi, the growing apical part of the hyphae is the major zone through which proteins are exported (Chang & Trevithick 1974; Wessels 1990). This has been confirmed by immunolabelling and micro-autoradiography of Aspergillus niger secreting glucoamylase (Wosten et al. 1991). It has been suggested that there are abnormally permeable sites in the cell walls of yeasts, through which there is a regulated exchange of macromolecules between the intracellular and extracellular media (Tsiomenko et al. 1987).

The authors are with the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292, Pushchino, Moscow Region, Russia; fax: (095) 923-36-02. ‘Corresponding author. 0

1994 Rapid Communications

lipase, molecular

forms, secretion.

In the present study the lipases secreted by the fungus Rhizopus microsporus are localized and lipase-containing structures that apparently cross the cell wall are demonstrated in the mycelium after Ca-alginate immobilization.

Materials and Methods Organisms and Culture Conditions microsporus UzLT-4B and UzLT-5C, from the Culture Collection, Institutue of Microbiology, Uzbekistan Academy of Sciences, Tashkent, were maintained on wort agar. For the experiments the strains were in 750 ml flasks, each with 100 ml of growth medium, shaken at 150 rev/min for 20 to 26 hat 37°C. The growth medium comprised 1% (w/v) yeast autolysate and 1% (v/v) sunflower seed oil in tap water. R&opus

Growth Estimation Growth was estimated by measuring the weight of washed cells after drying at 105’C to constant weight. Preparation of Extracellular Lipases The culture broth was separated from the mycelia by filtration and then centrifuged at 6OOOxg for 15min at 4°C. The supematant was used as the extracellular lipase fraction (Diyorov et al. 1993a).

of Oxford Ltd

World @UIUI ojMicrobiology &Biotechnology, Vol IO, 1994

367

J. K. Diyorov

et

al.

Preparation of Intracellular Lipases After cultivation the mycelia were collected by filtration, washed with distilled water and resuspended in 0.05 M phosphate buffer, pH 7.5. The mycelia were then disrupted in a mortar with quartz sand, held in an ice bath. Cell debris was removed by centrifugation at 6000 x g for 15 min at 4V, and the resulting cellfree extract was used as the intracellular lipase fraction (Diyorov et al. 1993a).

Purification of Lipase Preparation of the homogeneous enzyme, using hydrophobic chromatography, gel filtration on Sephadex G-100 and FPLC on a Mono Q column was as described previously (Diyorov et al. 1993a,b).

Assay of Lipase Activity The activity of lipase was 1991), using a 40% (v/v) (v/v) polyvinyl alcohol, llpase was defined as the fatty acid/min.

measured in a pH-stat (Davranov et al. emulsion of olive oil stabilized with 2% pH 8.0, as substrate. One unit (W of amount of enzyme that liberated 1~01

Preparation of Polyclonal Antibodies Antiserum against lipase was obtained by immunizing rabbits with two of the purified enzymes (one of 45 kDa from strain UzLT-4B and one of 66 kDa from strain UzLT-50, using Freund’s complete adjuvant. The enzyme, in sterile 0.15~ NaCl, was mixed with an equal amount of the adjuvant before subcutaneous injection. Each rabbit was given another three injections over 2 weeks, using Freund’s incomplete adjuvant. Blood tests were performed 19 days after the last injection. Serum titres were determined by double diffusion in agar gel. The serum globulin fraction was extracted by double fractionation with ammonium sulphate, dialysed against 0.01 M phosphate buffer, pH 7.5, and separated by anion-exchange chromatography on a column with DE-52 cellulose (Whatman). The IgG-containing fractions were combined and concentrated on an UM-10 ultrafilter.

without antibodies were stained with (4 min, 24°C) and (Jeol).

or with pre-immune rabbit serum. Sections uranyl acetate (20 min, 37°C) and lead citrate examined in a JEM-100B electron microscope

Immobilization in Ca-alginate Gel Freshly-harvested mycelium (3 g) was mixed with 6 ml of 2.5% (w/v) sodium alginate in sterile 0.15 M NaCl and the mixture pipetted into 0.1 M CaCI,. Gel granules with incorporated mycelium were stabilized in the CaCl, solution for 0.5 hand then washed in the cultivation medium. Immobilization was performed under sterile conditions. The immobilized cells were then incubated in growth medium as described earlier.

Results strains of Rh. microsporus exhibited intracellular and extracellular lipolytic activity at the onset of growth. This activity increased and achieved its maximum in the early stationary phase (Figures 1 and 2). Strain UzLT-5C had five lipases of 24,32,43,66 and 98 kDa, three of which (32,66 and 98 kDa) were found both intra- and extra-cellularly. The other two lipases (24 and 43 kDa) were Both

Electrophoresis and lmmunoblotting Techniques SDS-PAGE and native electrophoresis were performed in 10% (w/v) gels according to Laemmli (1970). Gels were stained with Coomasie R-250, as described by Gorg et al. (1987). Proteins separated by SDS electrophoresis were transferred to OZ-pm pore Biodyne nitrocellulose sheets using a Transfer unit (LKBPharmacia) (Towbin et al. 1979).

Immune-electron Microscopy The mycelia of Rh. microsporus UzLT-5C

and UZLT-~B, grown in liquid medium to maximal stages of extracellular lipase activity (i.e. for 18 and 24 h, respectively) and that of Rh. microsporus UzLT-5C immobilized in Ca-alginate gel and incubated in a liquid medium for 40 h, were examined. Pellets of the free cells and granules of the immobilized fungi were resuspended in 0.05 M cacodylate buffer (pH 7.2) containing 2% glutaraldehyde at 4°C. After fixation (2 h) the samples were dehydrated in an ethanol series and embedded in Lowicryl K4 M low-temperature resin (Carlemalm et al. 1982). Ultrathin sections, cut with a diamond knife, were mounted on nickel grids covered with formvar film. Immunocytochemical staining, using the polyclonal rabbit antibodies at 1: 100 to 1: 1000 and protein A-gold complexes (15nm particles; Serva), was performed as described by Roth (1982). The specificity of the immunocytochemical reaction to lipase was tested by incubating the sections with protein A-gold

368

World @mud

ofMicrobiology

& Biotechnology, Vol 10.1994

01

&I

6

$1

$0

1

10

1

”

11

6 10 12 14 16 16 20 22 14 26 26 30 32 34 36 36 40 42’

Cultivation

Figure

1. Changes in extracellular lipase activity during growth of l -Mycelium dry weight.

time (h) (0) and

intracellular (+) UzLT-5C.

Rh. microsporus

“SIB

6

Figure

3 10 12 14 16 I8 20 22 24 20 28 30 32 34 36 38 40 420 Cultivation time (h)

2. Changes in extracellular lipase activity during growth of l -Mycelium dry weight.

(0) and

intracellular (+) UzLT-4B.

Rh. microsporus

Rhizopus microsporus lipases

Figure 5. lmmunoblotting of secreted Rh. microsporus UzLT-5C lipases during growth. 1-10 h; 2-12 h; 3-14 h; 4-16 h; 5-16 h; 6-24 h. Figure 3. SDS electrophoresis of purified Rh. microsporus UzLT-5C lipase preparations in 10% polyacrylamide gel. 1Marker proteins; 2--extracelluar lipase (32 kDa); 3extracellular lipase (66kDa); 4--extracellular lipase (98 kDa); 5-intracellular lipase (24 kDa); Gjntracellular lipase (43 kDa).

only found in the mycelial homogenate. All five forms were purified to homogeneity (Figure 3). Strain UzLT-4B had two extracellular (45 and 69 kDa) and two intracellular (28 and 40 kDa) lipases (Figure 4). All the lipases studied were monomers, since their apparent Mr were identical after separation in native and denaturing conditions (data not shown). Using irnmunoblot analysis, strain UzLT-5C was shown to secrete one enzyme (66 kDa) in the growth phase and the

other two secreted lipases (32 and 98 kDa) in the stationary phase (Figure 5). Strain UzLT4B secreted the 69 kDa lipase in the growth phase, while the other form (45 kDa) appeared in the stationary phase (Figure 6). Antibodies were obtained against the 66 and 45 kDa lipases from strains UzLT-5C and UzLT-4B, respectively. However, all the other molecular forms of lipase cross-reacted with these antibodies. Electron microscopy of ultrathin sections of both strains after growth in a liquid medium with sunflower seed oil revealed that anti-lipase antisera bound mostly to the cell envelope and to vesicles 0.3 to 1 pm in diameter located near the plasmalemma (Figure 7A and 8).These vesicles were seen to fuse with the periplasmic space (Figure 7B). UzLT-5C hyphae often had the immunolabel discretely distributed along the perimeter of the periplasmic space, although large areas of the periplasm had no immuno-label (Figure 7D). Intensive labelling of the growing tip of the hyphae was sometimes observed (Figure 7C). Immobilized hyphae were seen to have lipase in rounded bodies and tubules (approx. 0.2 pm in diameter) filled with an electron-dense substance and localized close to the

Figure 4. SDS-electrophoresis of purified Rh. microsporus UzLT-4B lipase in 10% polyacrylamide gel. 1-Marker proteins; 2-intracellular lipase (45 kDa); 3-extracellular (28 kDah 5lipase (69kDah 4--intm~4ular lipintracellular lipase (40 kDa).

Figure 6. lrnmunoblotting of secreted Rh. microsporus4B lipases during growth. 1-16 h; 2-20 h; 3-24 h.

World Journal of Microbiology b Biotechnology, Vol 10,1994

369

J. K. Diyorov et al.

Figure 7. Electron micrographs of the free mycelium ultrastructure. A. lmmunogold labelling of lipases in Rh. microsporus UzLT-4B. Note the uniform distribution of gold label in the periplasm. Arrows indicate the secretion vesicles near the plasma membrane. N-nucleus, M-mitochondria, V-vacuole. Scale bar: 1 pm. B. Electron-lightvesicles with immunogold labels. Bar: 0.2 pm. C and D. lmmunogold labelling of lipases in Rh. microsporus UzLT-5C. Note the uneven distribution of gold label all over the periplasmic space. Scale bar: 0.2 pm. plasmalemma (Figure 8A and B). Since the diameter of the rounded bodies coincided with that of the tubules, the bodies might be transverse sections of tubules. Immobilized mycelium incubated for 15 to 20 h featured unusual structures in the cell envelope that have not been

370

Wo~IdJovrnal ofMict.obiology & Biotechnology, Vol10,1994

described before. The structures were electron-dense formations that appeared to pierce the cell wall (Figures 8C to F). These structures accumulated a large amount of the immunogold-anti-lipase label. The tubules and rounded bodies filled with electron-dense substance are probably sections

Rhizopus microsporus lipases

Figure 8. lmmunogold labelling of Rh, microsporus UzLT-5C sections immobilized in Ca-alginate gel with anti-lipase. (A and 8):Rounded bodies and tubules with electron-dense substance. (C, D, E, F): lmmunogold labelling of "lipase channels" in the sections of immobilized Rh. micr~sporusUzLT-5C after 20 h incubation. Scale bars: 0.2 Fm.

World lournal of Microbiology b Biotechnology, Vol10,1994

371

J. K. Diyorov

et al.

of these structures, being similar in electron-density and immunolabel concentration. When the immobilized cells were incubated for 30 h the immunolabel was found outside the cell (data not shown).

Discussion Since detailed information about the methods used to isolate and purify both intra- and extra-cellular lipases has been published previously (Diyorov et al. 1993 a,b), the present study concentrated on cytological studies of lipase secretion. The electron microscopy shows both strains to have a systern of peripheral vesicles which stain with the immunogold anti-lipase label. Those vesicles closely associated with the periplasm (Figure 7B) are probably secretory and the rest are probably vacuoles. Some immunolabel was found in the cell wall itself, though the bulk was localized in the periplasm (Figure 7A and D). These observations confirm the intracellular localization of lipases indicated by differential centrifugation (Davranov et al. 1980). The heterogeneous distribution of lipase along the perimeter of the cell envelope of UzLT-5C, with extensive peripheral zones devoid of the immunolabel (Figure 7C and D), may be indicative of preferential protein secretion in the growing hyphal tip, as observed in various filamentous fungi (Chang & Trevithick 1974; Wessels 1990). The structures filled with anti-lipase label and located in the region of the Rh. microsporus UzLT-5C cell envelope and the cytoplasm adjacent to it (Figure 8C to F) are probably specialized sites of lipase export. As these structures have never been observed before and were only found in one fungal strain after its immobilization and at a definite phase of hyphal develop ment (E-20 h after immobilization), they may be ‘lipase channels’ that are formed and reformed at a high rate. Immobilization of the mycelium into a dense gel matrix may inhibit diffusion of secreted lipases through the cell wall and, consequently, help to retain them in the lipase channels. The existence of specialized sites in the cell wall with abnormal permeability for high-molecular-weight proteins has been predicted (Tsiomenko et al. 1987; Lupashin et al. 1992). However, since cytology has failed to detect the structures that perform this function in lower eukaryotes, these structures are probably unstable. They must appear and disappear rapidly, making detection by electron microscopy difficult. A characteristic feature of various microbial lipases is their occurrence in several molecular forms (Iwai & Tsujisaka 1984); Rh. microsporus is obviously no exception. The molecular forms of lipases in most fungal species and strains are immunologically identical (Yamaguchi & Mase 1991). However, Shimada et al. (1990) showed that, in the fungus Geotrichum candidum, different genes code for two forms of lipase differing in amino acid sequences. Polyclonal antibodies obtained for each lipase form were specific (Charton et 372

World Journal

of Microbiology

6 Biotechnology, Vol lo,1994

al. 19921, enabling each particular form of lipase in the fungal cells to be localized immunocytochemically. This was impossible in the present study, since all lipase forms of Rh. microsporus cross-reacted with antibodies produced against the 45 and 66 kDa forms. The present biochemical and cytological data indicate that export of at least some of the molecular forms of lipase in Rh. microsporus is realized by a mechanism distinct from the constitutive secretory pathway proposed for yeasts (Schekman 1985). In Rh. microsporus, exported lipase forms probably exit the cell via a special route in which channels crossing the cell wall might play a crucial role. Further studies on the phenomenon of lipase channels, including how they function, their ultrastructure and the chemical nature of their electron-dense contents are required. The timing and conditions of their formation and reformation in the cell wall need to be elucidated in Rh. microsporus and, probably, other fungi.

References Carlemalm, E., Saravito, R.M & Viliger, W. 1982 Resin development for electron microscopy and analysis of embedding at low temperature. Journal ofMicroscopy 126,123-143. Chang, P.L.Y. & Trevithick, J.R. 1974 How important is secretion of exoenzymes through apical cell walls of fungi? Archives of Microbiology 101, 281-293. Charton, E., Davies, C. & Macrae, A.R. 1992 Use of specific polyclonal antibodies to detect heterogeneous lipases from Geotrichum candidum. Biochimica et Biophysicu Actu 1127,191198.

Davranov, K.D., Alimdzhanova, M.I. & Bezborodov, A.M. 1980 Lipase activity of Oospora la&s protoplasts. Mikrobiologiya 49,421-426. Davranov, K.D., Sattarov, A. & Diyorov, J.K. 1991 Immobilized Oospora la&is lipase preparations and their properties. Collection of Czechoslovak Chemical Communications, 56,499504.

Diyorov, J.K., Tsiomenko, A.B., Davranov, K.D. & Kulaev, I.S. 1993a Hydrophobic chromatography and characterization of lipases secreted by fungus Rhizopus microsporus YzLT4B. Biokhimiya 58,979-986. Diyorov, J.K., Tsiomenko, A.B., Davranov, K.D. & Kulaev, IS. 1993b Purification and properties of intracellular lipase of fungus Rhizopus microsporus. Biotechnology (Russ.) 7,26-30. Gorg, A., Postel, W. & Weser, J. 1987 Horizontal SDS electrophoresis in ultrathin pore-gradient gels for analysis of urinary protein. Science Tools 32, 5-9. Huge-Jensen, B., Galluzzo, D.R. & Jensen, R.G. 1987 Partial purification and characterization of free and immobilized lipases from Mucor miehei. Lipids 22,559-565. Iwai, M. & Tsujisaka, Y. 1984 Fungal lipase. In Lipases, eds Borgstrom, B. & Brockman, H.L. pp. 443-469, Amsterdam: Elsevier Science.

Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680685. Lupashin,

V.V.,

Kononova,

S.V.,

Ratner,

Y.N.,

Tsiomenko,

Rhizopus microsporus lipases A.B. & Kulaev, IS. 1992 Identification of a novel secreted glycoprotein of the yeast Saccharomyces cerevisiae stimulated by heat shock. Yeast 8,157-169. Meachum, E.D., Colvin, H.J. & Braymer, H.D. 1971 Chemical and physical studies of Neurospora crassa invertase. Molecular weight, amino acid and carbohydrate composition, and quaternary structure. Biochemistry 10,326-332. Money, N.P. 1990 Measurement of pore size in the hyphae cell wall of Achlya bisexualis. Experimental Mycology 14,234-242. Rivera-Munoz, G., Tinoco-Valencia, J.R., Sanchez, S. & Farres, A. 1991 Production of microbial lipases in a solid state fermentation system. Biotechnology Letters 13,277-280. Roth, J. 1982 The protein A-gold (pAg) technique. Qualitative and quantitative approach for antigen localization on thin sections. In Techniques in Immunocytochemistry, Vol. 1, eds Bullock, G.R. & Petrusz, I’. pp. 107-133, London: Academic Press. Schekman, R. 1985 Protein localization and membrane traffic in yeast. Annual Review of Cell Biology 1,115-143. Shimada, Y., Sugihara, A., Iizumi, T. & Tominaga, Y. 1990 cDNA cloning and characterization of Geofrichum candidum lipase II. Journal of Biochemistry 107,703-707. Towbin, H., Stachelin, T. & Gordon, J. 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellu-

lose sheets: procedure and some applications. Proceeding of the National Academy of Sciences of the USA 76,4350-4354. Trevithick, J.R. & Metzenberg, R.L. 1966 Genetic alteration of pore size and other properties of the Neurospora cell wall. Journal of Bacteriology 92,10X-1020. Tsiomenko, A.B., Lupashin, V.V., Dmitriyev, V.V. & Kulaev, I.S. 1987 Cell wall permeability and protein export into the cultural broth in Saccharomyces cerevisiae. Mihrobiologiya 56,

797-804. Wessels, J.G.H. 1990 Role of cell wall architecture in fungal tip growth generation. In Tip Growth in Plant and Fungal Walls, ed Heath LB. pp. l-29. San Diego: Academic Press. Wosten, H.A.B., Moukha, S.M., Sietsma, J.H. & Wessels, J.G.H. 1991 Localization of growth and secretion of proteins in Aspergillus niger. Journal of General Microbiology 137, 2017-

2023. Yamaguchi, S. & Mase, T. 1991. Purification and characterization of monoand diacylglycerol lipase isolated from Penicillium camembertii U-150. Applied Microbiology and Biotechnology 34,720-725.

(Received accepted

in revised 2.5 November

form 8 November

1993;

2993)

World Journal of Microbiology & Biotechnology, Vol 10, 1994

373