E. Galas

Microbial β-Glucanases Different from Cellulases

AbstractThe β-glucans different from cellulose are the most abundant class of polysaccharides. They are found in microorganisms and higher plants as structural entities of cell wall, as cytoplasmic and vacuolar reserve materials, and as extracellular substances. Enzyme systems capable to hydrolyze β-glucans are produced by different microorganisms. The occurrence and nature of β-glucanases and their substrates are reviewed. The regulation of biosynthesis of these enzymes, their properties, substrate and product specificities, mode of action and molecular cloning are described. The participation of β-glucanases in the morphogenetic events of yeast cell is presented. The role and synergism of different types of 1,3-β-glucanases in microbial cell wall lysis and the potential application for isolation of intracellular materials like proteins, carbohydrates, enzymes and as an analytical tool are discussed in the light of current knowledge.

Enzymatic lactonization of 15-hydroxypentadecanoic and 16-hydroxyhexadecanoic acids to macrocyclic lactones

Abstract It has been demonstrated that Mucor javanicus L46 and Mucor miehei catalyse the lactonization reaction of 15-hydroxypentadecanoic and 16-hydroxyhexadecanoic acids to appropriate macrocyclic mono- and oligolactones. It has been found that petroleum ether and toluene and their mixture having the polarity log P = 2.96 form an advantageous environment for the reaction. The highest efficiency of synthesis is for toluene at 80°C and for petroleum ether at 70°C. The pH of the essential water layer, as well as the addition of pyridine or DMF, exerts an apparent influence on the reaction rate. The yield of monolactone synthesis is from a few percent to over 30%.

Two-step mutagenesis of Pullularia pullulans leading to clones producing pure pullulan with high yield

A wild strain of Pullularia pullulans, producing, under test conditions, 3 g l-1 of exopolysaccharide (pullulan) contaminated with melanin pigments, was mutagenized by UV irradiation to yield a clone producing white, uncolored product. UV irradiation of this clone gave new clones producing pure pullulan with high yield (about 10 g l-1 under test conditions and about 70 g l-1 under optimized conditions).

1,3-β-Glucanases fromStreptomyces sp.1228 lytic enzyme system

SummaryFour 1,3-β-glucanases GI, GII, GIV and GVIII from a culture filtrate ofStreptomyces sp. 1228 were purified by anion exchange chromatography using DEAE-Sepharose Cl-6B or DEAE-Cellulose, gel filtration on Bio-Gel P-200 or Sephacryl S-200, Amicon ultrafiltration and preparative PAGE. The Mr of these enzymes were 19000, 74000, 78000 and 56000 respectively. The glucanase GVIII consisted of two subunits. The optimal catalytic activity of the purified preparations was at 50–55°C and pH 5.5–6.0. The enzymes were also most stable at this pH. Both glucanases GI and GVIII were characterized by high thermostability. The glucanases showed different affinities towards laminarin with Km values of 6.65 x 10−5 mol/l for GI, 2.35 x 10−4 mol/l for GII, 8.1 x 10−5mol/l for GIV and 8.1 x 10−4mol/l for GVIII. The presence of metal ions was not required for activity of these enzymes but thiol groups increased their activity. D-glucono-δ-lactone did not inhibit the enzymes.

Mathematical modelling of ester synthesis by lipase in biphasic system

Abstract The invented mathematical model of ester synthesis in a biphasic system using the immobilised Mucor circinelloides lipase, situated in organic phase, was experimentally verified by butyl oleate, oleyl oleate, sucrose oleate and sucrose caprylate synthesis. The model took into consideration the fact revealed in experiments that water being a by-product of a reaction, created a separate phase thus influencing a value of equilibrium constant in the organic phase (KO). The applied mathematical model makes it possible to determine with high accuracy the yield of ester synthesis that can be obtained in the reaction poststational state.

Optimization of productivity of pullulan by means of multivariable linear regression analysis

Abstract Biosynthesis of pullulan by a mutant clone of Pullularia pullulans (producing polysaccharide not contaminated with melanins) was optimized by means of finding numerically, using multivariable linear regression analysis, the maximum of an arbitrarily chosen second-order polynomial model function of several variables which describe the dependence of the pullulan productivity (expressed in wt. % of the fermentation broth) on medium component concentrations and pH. The determined optimum medium composition for a 4-day fermentation at 28°C: sucrose, 35.3; NH 4 NO 3 , 0.031; KNO 3 , 0.092; MgSO 4 · 7H 2 O, 0.023; K 2 HPO 4 , 0.069; FeSO 4 · 7H 2 O, 0.0035; yeast extract, 0.031 (all in wt. %), initial pH 5.56, allowed to obtain 12.2 wt. % of pullulan (3.5 times higher than before optimization).

PVA-biocatalyst with entrapped viable Bacillus subtilis cells

Abstract Bacillus subtilis cells were entrapped in polyvinyl alcohol (PVA)-cryogel beads without decay in their viability and capability of secretion of proteolytic enzymes (metalloproteinase and subtilisin). Conditions for preparation of the PVA-biocatalyst with suitable stability and viability of B. subtilis cells were optimized. Diffusion of various compounds into the cryogel (sliced beads) has been monitored on-line using image analysis system. Optimal working conditions and kinetic constants for hydrolysis of proteins catalyzed by the PVA-biocatalyst containing whole B. subtilis cells were estimated. The PVA-biocatalyst was applied in the hydrolysis of casein. The productivity of the biocatalyst (expressed as an amount of liberated aromatic amino acids) reached a maximal level of 12 mg g−1 h−1. Composition of mixture of peptides was dependent on pH, concentrations of Na+ and glucose, and in the reaction milieu. Protein hydrolysates of desired composition can be obtained using B. subtilis viable cells immobilized in PVA-gel. Incubation of the immobilized cells in a nutrient medium with casein successfully regenerated proteolytic activity of the biocatalyst.

Activity of immobilised in situ intracellular lipases from Mucor circinelloides and Mucor racemosus in the synthesis of sucrose esters

The activity of intracellular, immobilised in situ lipases from Mucor circinelloides and Mucor racemosus can be changed by means of chemical modifications of the reaction milieu, using some substances isolated from Mucor cells. The substances act ambivalently (as activators or inhibitors) on the lipases. The yield of sucrose monocaprylate synthesis and the time to reach the reaction equilibrium state were determined in mono- and biphasic systems. The investigations proved that in a milieu of di-n-pentyl ether saturated with water, 92% of sucrose was esterificated, and the location of the lipase on the interface between the phases, markedly diminished the time equilibrium to reach.

Enzymatic isomaltooligosaccharides production

The subject of our study was the enzymatic synthesis of isomaltooligosaccharides (IMOs) from sucrose using two enzymes produced in our laboratory, i.e. dextransucrase (DS) from Leuconostoc mesenteroides and dextranase (D) from Penicillium funiculosum. Dextransucrease converts sucrose into dextran with fructose (Fru) as a by-product, whereas dextranase transforms dextran into IMOs. The research was focused on elucidation of the effect of, reaction time and enzyme and sucrose concentration, as well as the on the IMOs production. The reactions were carried out for 24 and 48 h, at 30°C and pH 5.4, in solutions containing different amounts of sucrose (5, 10, 20 and 40% w/v). It was shown that the composition of reaction products in particular depended on the concentration of sucrose solutions. The highest yield of isomaltose (IM), about 50% of total determined sugars, was achieved in 10 and 20% sucrose solutions. In the 10% case, glucose (Glc) predominated among the other reaction products, while it was isomaltotriose (IM3) at 20%. At higher sucrose concentrations even more IM3, and leucrose (Leucr), were formed.

Specificity of 1,3-β-glucanases from the lytic system ofStreptomyces sp. 1228

SummaryFour 1,3-β-glucanases GI, GII, GIV and GVIII, which were isolated from culture filtrate ofStreptomyces sp. 1228, were examined for their activities on different oligo- and polysaccharides containing 1,3-β-and/or 1,4-β-and/or 1,6-β-glycosodic bonds. Linear 1,3-β-glucan-laminarin was extensively degraded. CM-cellulose, xylan, lichenan and yeast glucan were hydrolyzed to different extents by the four enzymes. No activity against barley glucan was found. Laminarioligosaccharides, with a higher degreeol polymerization were more extensively hydrolyzed by GI, GII, and GIV enzymes. Glucanase GVIII prefers substrates with lower molecular mass.