Synthesis of extracellular (1 → 3)- and (1 → 6)-β-glucanase activities in carbon limiting chemostat culture by the fungus Acremonium sp. IMI 383068

When Acremonium sp. IMI 383068 was grown under chemostat conditions with scleroglucan as sole carbon source, the fungus produced four (1 → 3)-β-glucanases and a single (1 → 6)-β-glucanase. Production of the (1 → 3)-β-glucanase was growth rate related while the (1 → 6)-β-glucanase was growth rate unrelated. Specific activities of both were affected by increasing agitation speed, independently of dissolved oxygen tension (DOT) or growth rates, falling at the higher speeds examined. They also increased as DOT levels increased. No evidence was obtained to substantiate any relationship between enzyme specific activities and hyphal branching frequencies.     

Inulinase (β-fructofuranosidase) production by Kluyeromyces marxianus in batch culture

Summary Production of inulinase in batch fermentation using various carbon sources with Kluyermoyces marxianus was examined. Inulinase synthesis in the culture proceeded parallel to cell growth. Glucose, fructose and sucrose were inferior carbon sources for inulinase broduction. Highest production (212 U/mL) was achieved on inulin based media.     

TEMPO-mediated oxidation of (1 → 3)-β-d-glucans

The 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation was applied to water-insoluble (1 → 3)-β-d-glucans, paramylon and curdlan, to prepare water-soluble oxidized products. When the addition level of NaClO used as the primary oxidant was 15 mmol per gram of the polysaccharide in the combination with catalytic amounts of TEMPO and NaBr under aqueous conditions at pH 10, water-soluble TEMPO-oxidized products were obtained quantitatively within 100 min. ¹³C NMR analysis of the TEMPO-oxidized products revealed that the C6 primary hydroxyl groups of both paramylon and curdlan were completely converted to carboxylate groups by the oxidation. Thus, new (1 → 3)-β-d-polyglucuronic acid sodium salts having almost homogeneous chemical structures can be obtained. The highly crystalline paramylon took longer time for the complete oxidation of the C6 primary hydroxyls to carboxylate groups than curdlan. However, remarkable depolymerization occurs on main chains during the oxidation, and the degrees of polymerization of the water-soluble TEMPO-oxidized products prepared from paramylon and curdlan were only 68 and 86, respectively.     

Glucomannan and branched (1 → 3)(1 → 6) β-glucan from the aposymbiotically grown Physcia kalbii mycobiont

Cultures of the mycobiont Physcia kalbii were obtained from germinated ascospores and cultivated on Sabouraud-Sucrose-agar medium. Alkaline extraction of freeze-dried mycelia provided a branched (1 → 3),(1 → 6)-β-glucan and a glucomannan, whose chemical structure was determined by monosaccharide composition, methylation, controlled Smith degradation and NMR spectroscopic analysis. The β-glucan had a (1 → 3)-linked β-glucopyranosyl backbone, partially substituted (approx. 50% of the units) at O-6. The side chains were formed by 6-O- (∼82%) and 2,6-O-linked-β-Glcp units, while the non-reducing ends were formed by β-glucopyranosyl residues. The glucomannan had (1 → 6)-linked α-Manp units in the main chain, almost all being substituted at O-2 by α-Manp and α-Glcp units. This glucomannan could be a typical polysaccharide of lichens from the family Physciaceae.     

Enzymatic degradation of amylouronate (α-(1 → 4)-linked glucuronan) by α-glucuronidase from Paenibacillus sp. TH501b

Enzymatic degradation of amylouronate (α-(1 → 4)-linked polyglucuronic acid sodium salt, α-(1 → 4)-linked glucuronan), which was prepared from water-soluble starch by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation, was investigated. A bacterial strain TH501b capable of degrading amylouronate was isolated from soil samples collected in the natural environment. Molecular analysis of the 16S rRNA gene showed that TH501b belongs to the genus Paenibacillus. A hydrolytic enzyme responsible for the degradation of amylouronate, amylouronate hydrolase-I (AUH-I), was detected in the cell-free extract of TH501b. AUH-I was purified by four steps of column chromatography and some properties were characterized. The molecular mass of the native AUH-I was estimated to be approximately 115 kDa by size exclusion chromatography (SEC), whereas sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) showed two major bands at 80 kDa and 46 kDa, respectively. The enzyme was most active at pH 6.0–7.0 and 30 °C. The SEC analysis of reaction products revealed that AUH-I liberated glucuronate as a sole product from amylouronate, indicating that AUH-I hydrolyzed amylouronate exolytically, and thus, was classified as α-glucuronidase.     

The effect of dissolved oxygen tension on 11β- and 19-hydroxylation of Reichstein's Substance S by Pellicularia filamentosa

Summary The 11- and 19-hydroxylation enzyme(s) of Pellicularia filamentosa IFO 6298 have been shown to be inducible by Reichstein's Substance S. By using the protein synthesis inhibitor, cycloheximide, in fermenter culture the effects of dissolved oxygen tension (DOT) on enzyme induction and enzyme expression have been separately investigated. For both hydroxylations, an optimum DOT for induction has been shown at 15% of saturation, while the optimum for expression is at 30% of saturation. The results have been verified in the absence of cycloheximide. Thus, maximum rates of hydroxylation are achieved when induction is performed at low DOT, followed by elevation to ensure maximum expression.     

Effect of glucose level on the batch production of β-1,3-glucanase by Trichoderma harzianum and cell growth

The effect of initial glucose concentration on g-1, 3-glucanase production by Trichoderma harzianum NCIM 1185 and cell growth was studied in a batch stirred tank bioreactor. The initial glucose level was varied between 5 g/dm³ and 100 g/dm³. A maximum g-1,3-glucanase production of 0.820 U was obtained using the fully optimized medium which had an initial glucose level of 8.63 g/dm³ and thereafter there was a steady decrease in g-1,3-glucanase production. No g-1,3-glucanase production was observed beyond initial glucose concentrations of 40 g/dm³ which suggests a possible catabolic repression on the enzyme synthesis. The inhibitory effect of increased initial glucose concentrations on cell growth has been studied and the Luong and the Han-Levenspiel models were used to explain the mechanism of inhibition.     

Kinetics and effect of nitrogen source feeding on production of poly-β-hydroxybutyric acid by fed-batch culture

Summary A kinetic study of the production of poly--hydroxybutyric acid (PHB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the PHB production phase. The effect of ammonia feeding on PHB production was consequently investigated. The nitrogen source (ammonia water) was supplied at a low constant feeding rate after the growth phase in which cell mass concentration reached 60 g/l. Feeding with a small quantity of ammonia resulted in a more rapid increase in intracellular PHB content than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated PHB but also reduction of microbial PHB synthetic activity.     

Optimized methodology for extraction of (1 → 3)(1 → 6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative

The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment.     

Optimization of uracil addition for curdlan (β-1→3-glucan) production by Agrobacterium sp.

Uracil, acting as a precursor of UDP-glucose, served as an activator on the production of curdlan with Agrobacterium sp. (ATCC 31750). The time of adding uracil was important to improve curdlan production. When uracil was added after ammonium depletion (at 26 h), it was used as a nitrogen source for cell growth. Although the cell concentration increased, the curdlan production was decreased. If uracil was added at 46 h, then uracil was degraded slowly but still activated curdlan production. With the addition of both sucrose (200 g) and uracil (1.5 g), the curdlan production was increased up to 93 g l^–1 after 160 h fermentation.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Effect of levulinic acid on cell growth and poly-β-hydroxyalkanoate production by Alcaligenes sp. SH-69

Summary Following growth of Alcaligenes sp. SH-69 on glucose as a sole carbon source for the production of poly--hydroxyalkanoates (PHAs), relatively low levels of levulinic acid (LA) were detected. Experiments were carried out in batch and continuous culture, and the effects of LA addition on growth and PHA synthesis were determined. Significant stimulatory effects were observed, greater than those for propionic acid addition. In N-limited two stage continuous culture, a maximal PHA content of 38.3 % (w/w) was achieved with a polyhydroxyvalerate (PHV) content of 23.5 % (molar basis) at D=0.078 l/h. This resulted from the controlled addition of LA at 0.5 g/L/h in the presence of excess glucose.     

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp,from rye arabinoxylan

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-l-arabinofuranosidase Axh43A indicated that U1 was α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp and U2 was α-l-Araf-(1 → 2)-d-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp from highly arabinosylated xylan.     

The investigation of effect of organic carbon sources addition in anaerobic–aerobic (low dissolved oxygen) sequencing batch reactor for nutrients removal from wastewaters

The effect of addition of organic carbon sources (acetic acid and waste activated sludge alkaline fermentation liquid) on anaerobic–aerobic (low dissolved oxygen, 0.15–0.45 mg/L) biological municipal wastewater treatment was investigated. The results showed that carbon source addition affected not only the transformations of polyhydroxyalkanoates (PHA), glycogen, nitrogen and phosphorus, but the net removal of nitrogen and phosphorus. The removal efficiencies of TN and TP were, respectively, 61% and 61% without organic carbon source addition, 81% and 95% with acetic acid addition, and 83% and 97% with waste activated sludge alkaline fermentation liquid addition. It seems that the alkaline fermentation liquid of waste biosolids generated in biological wastewater treatment plant can be used to replace acetic acid as an additional carbon source to improve the anaerobic–aerobic (low dissolved oxygen) municipal wastewater nutrients removal although its use was observed to cause a slight increase of effluent BOD and COD concentrations.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Different contributions of side-chains in β-d-(1 → 3,6)-galactans on intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge

Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer’s patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with β-d-(1 → 3)-galactan moieties branched with β-d-(1 → 6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the β-d-(1 → 3)-galactan or β-d-(1 → 6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with β-d-(1 → 3,6)-galactosyl side-chains having terminal β-d-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.     

Studies on β-glucanases. Some properties of a bacterial endo-β-(1→3)-glucanase system

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley β-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific β-glucanases, an endo-β-(1→3)-glucanase and an endo-β-(1→4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-β-(1→3)-glucanase having a molecular weight of about 16000.