SUMMARYSubmerged cultures of Claviceps fusiformis (Loveless) became progressively viscous during the incubation because of the production of an extracellular polysaccharide. The polysaccharide was shown to be a branched glucan with a PI + 3 linked main chain having single glucopyranosyl units at intervals along it in the PI + 6 configuration. For most of the media investigated growth at 27O and between pH 5 and 6 was accompanied by glucan production, which ceased when growth was arrested by lack of nutrient. The glucan was re-metabolized by the fungus only to a limited extent as the culture aged; the degree of branching varied during the culture period. The unit amount of glucan synthesized was not affected by numerous successive subculturings over a period of several months, but the product from the later cultures had a greater degree of branching. Branched glucans of similar structure to the above were also detected in low concentration in the natural sclerotia. The presence of PI -+ 3 linked glucans in the cell walls of this and other fungal strains examined was also indicated.

Evidence suggests that sucrose is the main carbon source for growth of Claviceps spp. in the parasitic condition. The sucrose acts as substrate for an active beta-fructofuranosidase, produced by the fungus, which in the first instance converts the disaccharide into glucose and an oligofructoside. In this way, 50% of the glucose, supplied as sucrose, is made available to the parasite for assimilation. Subsequent action of the enzyme on both sucrose and the oligofructoside leads to the release of more glucose and the formation of additional oligosaccharides. The structures of the main oligosaccharides formed have been elucidated and the interactions of each compound studied. In experiments with purified enzyme in vitro the interaction of the oligosaccharides is rapid but in culture they are assimilated only slowly; in each case some free fructose is liberated. Free fructose is not assimilated in the presence of glucose and, further, inhibits growth at concentrations which might be expected to occur in the parasitic condition. A dual role has been suggested for the enzyme, with sucrose as substrate, in which glucose is made available to the growing parasite, while at the same time transfer of the fructose to form oligosaccharides prevents it from accumulating at inhibitory concentrations. Ultimately, when glucose becomes limiting, the fungus will adapt to fructose assimilation.

S U M M A R YClaviceps fusiformis usually produces a stable viscous glucan during submerged culture fermentations. A new strain 139/2/1 G of the fungus, which subsequently autolysed this glucan to glucose, was studied and the autolysis ascribed to a constitutive PI -+ 3 glucanase and a P-glucosidase which were detected as soon as the fungal hyphae differentiated to a sclerotial form. The glucanase production followed a sigmoid pattern, reaching a maximum within 12 days, and the liberated glucose contributed to renewed growth towards the end of the fermentation. A sucrase and a maltase were also detected. Maximum glucan autolysis was achieved by using a large spore inoculum. This maintained minimal viscocity throughout the fermentation and, by maintaining adequate aeration, has since facilitated ergot alkaloid production by the organism. I N T R O D U C T I O NDavis, Rhodes & Shulke (1965) described the significant decrease of extracellular glucan in submerged cultures of Plectania occidentalis and a Helotium sp. towards the end of the fermentation and they presumed that this was due to the secretion of an ' exocellular glucanase ' by the organism. However, no autolysis was observed during studies on the structurally similar glucan produced by Claviceps fusiformis (Buck, Chen, Dickerson, & Chain, I 968). The production of alkaloids in submerged cultures by C. fusiformis is adversely affected by the formation of the viscous glucan, and during investigations on the alkaloid fermentation process a new variant strain (I 39/2/1 G ) arose spontaneously and was selected for its ability to autolyse the extracellular. glucan. The usual non-autolysing strains yielded a stable and highly viscous growth within 7 days, whereas the new variant autolysed completely after a further 7 to 10 days. In view of the structure of the extracellular glucan, its autolysis by strain 139/2/1 G has been investigated to determine whether this is due to the activity of glucanases similar to those reported by Reese & Mandels (1959) for a variety of fungi. sucrose + asparagine liquid medium was sterilized in 500 ml. Erlenmeyer flasks at 106" for 15 min. A modified medium with 10% mannitol (w/v) instead of the sucrose was also used. METHODS OriginSeed cultures. A spore suspension (2 to 6 x 109 spores/ml.) was prepared from 14-day agar cultures and I ml. inoculated into each 500 ml. flask. Usually the fermentation was completed without further transfer, but in some experiments the first (seed) stage flasks were subsequently used as a source of inoculum for a second stage. Where mycelial inocula were used an aqueous suspension was prepared by crushing the sparsely sporing mycelium from 3-day agar cultures.Flasks were incubated at 27' on a rotary shaker (Buck et al. 1968). For sampling, duplicate flasks were removed at intervals from the shaker and the course of fermentation was followed by the following parameters : microscopic examination and measurement of pH value, sugar utilization, nitrogen utilization, dry wejght of mycelium and polysaccharid...

Mannitol dehydrogenase (EC 1 . 1 . 1 .138) has been purified to homogeneity from fruit bodies of Agaricus bisporus. M , values of 115000 were determined by gel filtration and 130000 by rate zonal ultracentrifugation. The sedimentation coefficient is 6.5s. The native protein is composed of four subunits of M , 29000. The enzyme is specific for NADP, and shows low activity with sorbitol. Normal Michaelis-Menten kinetics are exhibited for both mannitol and NADP, giving K , values of 16-2 mM and 36 p~ respectively at pH 7.0. The K , value for NADPH is 38.5 p~ and that for fructose approximately 1.2 M. The V,,, is 591 p~ min-' (mg protein)-' for mannitol synthesis and 5 pmol min-I (mg protein)-' for fructose synthesis at pH 7.0. Inhibition of fructose synthesis by NADPH is stronger than inhibition of mannitol synthesis by NADP. The results are discussed with respect to the control of enzyme activity under physiological conditions.

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