Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr.

Protoplasts of the ectomycorrhizal ascomycete Cenococcum geophilum were isolated from mycelium grown in liquid medium. The method was optimized with regard to culture conditions, preincubation, lytic enzyme system, pH value of the incubation medium, osmotic buffer and incubation temperature for C. geophilum strains SIV and 1448. The yields were 1-3·10⁸ and 7·10⁶ protoplasts per gram fresh weight for C. geophilum SIV and C. geophilum 1448, respectively. Protoplasts from C. geophilum SIV exhibited plasma membrane integrity close to 100% (fluorescein diacetate staining). At least 50% of the protoplasts contained a nucleus (staining with acridine orange). The regeneration of protoplasts from C. geophilum is described for the first time. The regeneration frequency was up to 13%, and, dependent on the conditions of culture (liquid medium, agarose, agar), four types of regeneration patterns could be distinguished Regenerated protoplasts of C. geophilum were capable of forming mycorrhizas with spruce (Picea abies) seedlings.     

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10⁷/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO₄ (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.     

Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1F-fructosyltransferase

Summary Favourable reaction conditions for the enzymatic production of 1-kestose by sucrose-1^F-fructosyltransferase, SFT (EC 2.4.1.99) from Aspergillus phoenicis CBS 294.80 mycelium were established. The intracellular enzyme SFT works best at 60°C, exhibits a relatively high thermostability and possesses an alkaline pH optimum. An invertase also present in the mycelium of A. phoenicis possesses an acidic pH optimum. Consequently, around pH 8.0 sucrose is converted mainly to 1-kestose and nystose while fructose is only formed in relatively small amounts. Under optimal conditions (55° C, pH 8.0 and an initial sucrose concentration of 750 g 1^-1) a yield of about 300 g 1-kestose per 1.01 reaction mixture could be achieved after 8 h.Offprint requests to: J. A. M. van Balken     

Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis

Summary The ability of serveral strains of Streptomyces to degrade cell walls from Fusarium scirpi was tested by plating them on agar containing a cell wall preparation derived from the fungus. In this assay, S. tsusimaensis was most effective in producing a clear zone of lysis during growth on the opaque medium. This Streptomyce strain was subsequently grown in liquid culture containing cell walls as the sole carbon source and the exoenzymes were isolated from the culture broth. The enzyme preparation produces a clear zone of lysis when filled into wells in the cell wall agar and was used to prepare protoplasts from F. scirpi. The protoplast yield was 1x10⁹ protoplasts/ml of enzyme solution from 35 mg dry weight of Fusarium mycelium. Protoplasts could be regenerated at a frequency of up to 80%.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Mold

A mold which produced α-galactosidase and little invertase was isolated and identified as Mortierella vinacea. α-Galactosidase formation of the mold was induced by galactose, melibiose, raffinose and lactose. Among these inducers lactose showed the most stimulative effect. α-Galactosidase was produced by either Koji method or submerged culture method, but in the latter most α-galactosidase was found in the mycelium fraction.

Hydrolysis of raffinose in beet molasses was studied with the α-galactosidase in the mycelium fraction and about 80% of raffinose was found to be hydrolyzed by the enzyme preparation.     

CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae

Summary Various gene fusions between the arginine permease and invertase have been constructed in order to obtain information about whether part of the CAN1 gene product can induce secretion of biologically active invertase missing its own signal sequence. A construction containing 30 N-terminal amino acid residues of the CAN1 gene product fused to invertase was not secreted. When the CAN1 portion was elongated to 477 or 560 amino acid residues, secretion of the fusion proteins was observed. A fusion lacking 59 amino acids at the amino-terminal end of the arginine permease was also secreted. These results indicate that the amino-terminal end of the arginine permease is neither sufficient nor essential for membrane insertion; instead this enzyme should contain an internal targeting sequence facilitating secretion. Some general implications on the biosynthesis and topology of membrane proteins are also discussed as well as the homology with histidine permease.     

Transformation of steroids by fungal protoplasts

Summary Protoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the induced mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.     

Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function

Summary Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K _i value being ca. 4×10^–2 M and 3×10^–1M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.     

A novel method for immobilization of invertase from the thermophilic fungus, Thermomyces lanuginosus

An invertase from the thermophilic fungus, Thermomyces lanuginosus was immobilized on phenyl-Sepharose and its properties were studied. Between the soluble and immobilized forms of the invertase, there were not much difference in their optimum pH, K _M and V _max for sucrose. In contrast, the K _M and V _max for raffinose changed significantly. The optimum temperature for the immobilized invertase was lower by 10 C. The immobilized invertase showed remarkable stability at 50 C and was less sensitive to inhibition by metal ions. There was no leaching of the enzyme for at least a month when stored in the refrigerator. The method is novel and specific for the thermophilic invertase as a mesophilic invertase (from yeast) did not bind to phenyl-Sepharose.     

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.     

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Summary Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically ¹⁴C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.     

The genetics of a putative social trait in natural populations of yeast

The sharing of secreted invertase by yeast cells is a well‐established laboratory model for cooperation, but the only evidence that such cooperation occurs in nature is that the SUC loci, which encode invertase, vary in number and functionality. Genotypes that do not produce invertase can act as ‘cheats’ in laboratory experiments, growing on the glucose that is released when invertase producers, or ‘cooperators’, digest sucrose. However, genetic variation for invertase production might instead be explained by adaptation of different populations to different local availabilities of sucrose, the substrate for invertase. Here we find that 110 wild yeast strains isolated from natural habitats, and all contained a single SUC locus and produced invertase; none were ‘cheats’. The only genetic variants we found were three strains isolated instead from sucrose‐rich nectar, which produced higher levels of invertase from three additional SUC loci at their subtelomeres. We argue that the pattern of SUC gene variation is better explained by local adaptation than by social conflict.     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

Production,separation and purification of yeast invertase as a by-product of continuous ethanol fermentation

Summary A strain of Saccharomyces uvarum produced extracellular invertase in a chemostat reactor using a medium containing corn steep liquor and sugars. The production of yeast invertase increased with increase in corn steep liquor concentration. The production rate of invertase was maximal at a dilution rate of 0.75 h^–1. The production rate of invertase was found to be affected by the type of sugar substrate and fermentation temperature. The invertase in the crude broth could be purified by one-step DEAE chromatography. An 84% enzyme recovery with ninefold purification and 30-fold concentration could be achieved using this simple isolation procedure. Offsprint requests to: L. F. Chen     

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (<65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - Con A concanavalin A - Tris tris(hydroxymethyl)-amino-methane     

Mutant invertase proteins accumulate in the yeast endoplasmic reticulum

Summary Intercompartmental transport of secreted proteins in yeast was analysed using invertase mutants. Deletions and insertions at the BamHI (position +787) or the Asp718 (position +1159) sites of the SUC2 gene led to mutant proteins with different behaviour regarding secretion, localization and enzyme activity. The deletion mutants showed accumulation of core glycosylated material in the endoplasmic reticulum (ER) a decrease of secreted protein by 5%–30% and loss of enzyme activity. The secreted material was localized in the culture medium and not — as is normal for invertase-in the cell wall. No delay in transport from the Golgi to the cell surface was observed, indicating that the rate-limiting step for secretion is at the ER-Golgi stage. Two insertion mutants, pIPA and pIPB, retained enzyme activity. Mutant pIPB showed 10% secretion, while 60%–70% secretion was observed for pIPA. While the non-secreted material accumulated in the ER, the secreted material was present in the cell wall. The results suggest that the presence of structures incompatible with secretion leads to ER accumulation of mutated invertase.     

Sugar Transport into Storage Tubers of Stachys sieboldii Miq.: Evidence for Symplastic Unloading and Stachyose Uptake into Storage Vacuoles by an H+-Antiport Mechanism*

Following assimilation of ¹⁴CO₂ by leaves of Stachys sieboldii, ¹⁴C-stachyose is translocated into the tubers. Stachyose is accumulated and stored in the vacuoles of the pith parenchyma. Protoplasts and vacuoles were isolated and the uptake of sugars was examined. Uptake of sucrose and sucrosyl oligosaccharides of the raffinose family by protoplasts was very low compared to glucose. Transport parameters for glucose indicated a carrier mediated transport in the lower concentration range which was superimposed by diffusion at higher concentrations (> 10 mM). The very low sugar uptake by protoplasts and the sparse enzyme activities of stachyose synthase in the storage parenchyma as well as acid invertase and α-galactosidase in the cell walls indicated symplastic unloading of stachyose in the tubers. Experiments on ¹⁴C-stachyose uptake by isolated vacuoles confirmed previous observations by Keller (1992). Isolated vacuoles exhibited ATP and PP hydrolysis and were capable of generating a proton gradient across the tonoplast by a V-type H⁺-ATPase and H⁺-PPase. This was demonstrated by fluorescence quenching of quinacrine. Fluorescence could be restored by the addition of gramicidin and partly recovered by the addition of stachyose; mannitol, sorbitol and glucose had no effect. Fluorescence recovery depended on the concentration of stachyose and revealed saturation kinetics (K_m = 28 mM). Comparable results have been obtained with tonoplast vesicles by Greutert and Keller (1993). Experimental data presented here provide circumstantial evidence for symplastic unloading of stachyose in the tubers of Stachys sieboldii and demonstrate that the stachyose concentration in the cytoplasm of storage parenchyma cells is kept low by active stachyose transport into the vacuoles. The results suggest a stachyose/H⁺-antiport system.