No abstract

An extracellular pectin lyase (PNL) [poly-(methoxygalacturonide)lyase; EC 4.2.2.10] produced by Penicillium italicum CECT 2294 grown on a surface bran (natural medium) or in a submerged (synthetic medium) culture was investigated. Both culture filtrates showed macerating activity at low pH on cucumber, potato, and orange tissues. The physicochemical properties of the enzyme obtained from both culture methods were identical, as well as its catalytic properties, which were assayed by different methods. The molecular mass of the PNL obtained by gel filtration chromatography was 22 kDa; the isoelectric point was 8.6, as determined by chromatofocusing; and the enzyme was able to catalyze the eliminative cleavage of pectins with low (37%) and high (from 54 to 82%) degrees of esterification. The PNL produced in liquid medium showed a K,m for pectin (degree of esterification, 70%) of 3.2 mg/ml, and the optimum pH was 6.0 to 7.0. This enzyme was stable at 50°C and at pH 8.0. The ability of this PNL to macerate plant tissues in acidic environmental conditions, its stability at low pH and temperatures up to 50°C (thus preventing mesophilic microbial growth), and the absence of pectinesterase make this preparation useful for the food industry.

k Dm after SDS.electrophoresis in pol)'acr~,'lumide gradient ~,cls. attd the isoeleetric point w~ts li,6 as determined by isoelectric toe,using. Th~ optimum ptl (9.0), the Itillh pH .nd temperature stnhilitie~ the abilib' to deBt,de pectins from diffcrenl sources and whh a wide ranl~le ofde~recs ofesteritien.ti~n (rrt~m 37% Io B6,.~] as well .s lhe imp0rtanec of" this tl~+p¢ of biocalaly~ts in lhe food industry make tllhl enzyme an intere~tim[t mtbject of study.

Cell‐free extracts of nitrate‐grown as well as of ammonium‐grown cells of the filamentous non‐nitrogen‐fixing cyanobacterium Phormidium laminosum (strain OH‐1‐p.Cl1) showed detectable levels of both glutamine synthetase (GS, EC 6.3.1.2) and NADPH‐dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) activities. The GS level of nitrate‐grown cells was higher than that of ammonium‐grown cells, whereas the GDH level was higher in ammonium‐grown cells and depended on the external ammonium concentration. When nitrate‐grown cells were transferred to an ammonium‐containing medium, a decrease of GS and an increase of GDH specific activities occurred, even in the presence of nitrate. Conversely, when ammonia‐grown cells were transferred to a nitrate‐containing medium, an increase of GS and a decrease of GDH‐specific activities took place. Both these effects were inhibited by chloramphenicol and were probably mediated by de novo protein synthesis. When either cell type was transferred to a medium without nitrogen source, the specific activities of both enzymes increased. When nitrate‐grown cells were transferred to nitrate medium with L‐methionine‐DL‐sulphoximine (MSX) added, the specific activity of GDH also increased. Here we present some evidence that, under certain conditions of nitrogen availability, GDH would play a minor role in ammonium assimilation.

Soluble glutamine synthetase activity (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) was purified to electrophoretic homogeneity from the filamentous non-N2-fixing cyanobacterium Phormidium laminosum (OH-1-p.C1l) by using conventional purification procedures in the absence of stabilizing ligands. The pure enzyme showed a specific activity of 152 pumol of y-glutamylhydroxamate formed * min-(transferase activity), which corresponded to 4.4 pumol of P1 released min-' (biosynthetic activity). (20), photosynthetic bacteria (3), and a wide range of cyanobacteria (see reference 11 for a recent review). However, in addition to the GS-glutamate synthase cycle, glutamate dehydrogenase (EC 1.4.1.4) (16) and alanine dehydrogenase (EC 1.4.1.1) (24) may also contribute to ammonium assimilation in cyanobacteria, especially under certain nutritional conditions of nitrogen availability. Recently, an alternative route to GSglutamate synthase for the primary assimilation of ammonium in Anabaena sp. strain 1F, involving the participation of GS in collaboration with carbamylphosphate synthetase (EC 2.7.2.9), has been proposed (5). Consequently, GS is subject to intensive regulatory control.Although the GS isolated from bacterial sources are similar in a number of features, such as molecular size (about 600 kilodaltons [kDa]), subunit structure (dodecamers composed of identical subunits of about 50 kDa), and the requirement of divalent cations for activity, their regulatory properties appear to be quite different. For example, the enzyme from Escherichia coli (28) and several other gramnegative bacteria (13) is regulated by covalent modification through adenylylation-deadenylylation. In contrast, the GS from gram-positive bacteria is susceptible to feedback regu-* Corresponding author.lation by intracellular concentrations of adenine nucleotides, amino acids, and divalent cations rather than by covalent modification (17). However, in blue-green algae (cyanobacteria), the enzyme can be activated by dithiol compounds in vitro or by reduced thioredoxin (27). The cyanobacterial enzyme is not susceptible to regulation by covalent modification, and the availability of divalent cations and presence of feedback inhibitors (mainly Gly, L-Ala, and L-Ser) may play the dominant role in regulating GS in vivo (22,30). Moreover, the cyanobacterial enzyme shows cumulative inhibition with mixtures of inhibitors (i.e., AMP, L-Ser, and L-Asp), which indicates that there are independent binding sites on the enzyme for the inhibitors (30). The Anabaena flos-aquae GS was also found to be regulated by the energy charge of the cell, and cooperative inhibition was caused by CTP and any single nucleotide (18).GS from several species of the filamentous N2-fixing cyanobacterial genera Anabaena (18,23,25,29) and Nostoc (25) has been purified to electrophoretical homogeneity and characterized. Despite abundant information on the enzyme from N2-fixing cyanobacteria, only data regarding the GS from the unicellular non-N2-fixing cyanobacterium Anacystis nidu...