Chemical composition of agars from a newly reported Japanese agarophyte,Gracilariopsis lemaneiformis

The chemical composition of agars from Gracilariopsis lemaneiformis, newly reported from Japan, was investigated. Native agars were isolated by a sequential extraction of plants in water at 22 °C and 100 °C, and in boiling 20, 40 and 60° ethanol. Agars in each extract were analyzed by chemical methods, ¹H, ¹³C NMR; and IR spectroscopy. The highest yield of agar (total carbohydrate) was obtained from the 40° ethanol extract (55°). Highest sulfate content was attained in non-alkali treated agars extracted with hot water (4.81°, DS 0.2). The 3,6-anhydrogalactose content was highest in the 40° ethanol extract (36.1° in non-alkali treatment, 40.3° in alkali treatment). The highest methoxyl content (6.51°, DS 0.66) was obtained in the 60° ethanol extract. The G. lemaneiformis agar is composed of the biological precursor to agarobiose repeating units and agarobiose containing 6-O-methyl agarobiose and a small amount of 2-O-methyl-α-l-galactopyranose residues. Alkali treatment improved the chemical quality of the agar fractions, which was comparable with Japanese commercial agar and agarose.     

α-1,4-Glucan lyase,a new class of starch/glycogen-degrading enzyme

Antibodies have been raised against an -1,4-glucan lyase purified from the red alga Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik. Localization of -1,4-glucan lyase in ultra-thin sections of the red alga was performed using immunogold/transmission electron microscopy. The enzyme was found exclusively in the stroma of the chloroplasts of the algal cells, not in the cell wall, cytosol or around the cytosolic starch granules. Partial amino-acid sequences of the algal lyase, with a total length of 100 amino-acid residues, were obtained. No sequence homology was found with proteins and peptides of known sequences.This work was supported by grants from the Swedish Council for Planning and Coordination of Research (FRN), Carl Tryggers Foundation, and Hierta-Retzius Fund. We thank Ms Katrin Österlund and Anette Axén for their expert technical assistance with this work.     

Optimization of the yield and quality of agar from Gracilariopsis lemaneiformis (Gracilariales) from the Gulf of California using an alkaline treatment

The effect of several alkali treatments on the yield, gel strength, rheology, and chemical characteristics (quality) of the agar obtained from Gracilariopsis lemaneiformis from the Gulf of California was analyzed using different alkali concentrations, temperatures and treatment times. In the first stage of the experiment, all treatments lasted 60 min and the NaOH concentrations (2.5, 3.0, 4.0, 5.0, 6.0%) and temperature (80, 90, 100°C) varied. At constant time, temperature played the predominant role, promoting an increase in agar gel strength. Based on the best treatment conditions found (4% and 5% NaOH, and 90°C and 100°C temperature), in the second stage different treatment times (15, 30, 60, 90, 120 min) were used. Since agar yields were not significantly different among temperatures and times, the optimal conditions to obtain best quality agar were those providing the highest gel strength. Treatment time played an important role in increasing gel strength. Maximum gel strength (Nikan, 954 g cm⁻²) was obtained with 5% NaOH at 100°C after 90 min of treatment, though these conditions resulted in an agar yield reduction of 25.5% relative to native agar. This treatment proved to efficiently yield G. lemaneiformis agar that will meet the commercial quality requirements regarding gel strength, 3,6 anhydrogalactose and sulfate content, as well as rheology and hysteresis. Enrique Hernández-Garibay holds a CONACyT scholarship.     

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (K _s) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly--hydroxybutyrate. The storage of poly--hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µ _m ) the poly--hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO₂: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.     

Does storage time influence yield and agar properties in the tropical agarophyte Gracilaria cornea?

The agar yield and quality characteristics of Gracilaria cornea from Yucatán, Mexico, werestudied during 18 months of storage. Biomass wasstored at a temperature of 22.1 ± 0.9 ^°Cand humidity of 59.8±3.6%. The agar contentvaried erratically, but the average value waspractically constant over the storage period with an average of 20.1 ± 1.5±. Gel strength, gelling and meltingtemperatures were negatively affected by the totalstorage time. No significant changes were found duringthe first five months for these characteristics withmean values of 1134 ± 57 g cm^-2, 40.8 ±0.4 ^°C and 91.2 ± 0.9 ^°Crespectively. Agar degradation was evident after thefifth month and accounted for a 17± loss in gelstrength and 7± in gelling and meltingtemperatures. Nevertheless, gel strength valuesremained around 930 ± 23 g cm^-2 with nosignificant changes until the end of the storageperiod. The decrease in gel strength showed asignificant relationship with decrease in3,6-anhydrogalactose but not variation in sulphatecontent. This was probably due to agar hydrolysiscaused by enzymatic processes of endogenous and/ormicrobial origin. These results suggest that thetropical G. cornea had a similar resistance todegradation during storage to that observed for G. chilensis, a cold water species. Agar qualityand yield in G. cornea after one and a half yearof storage are within the range of food grade agar.     

Process optimization for the production of sugar for the bioethanol industry from Tapioca,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse tapioca powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 120 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 60 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of tapioca starch.     

Polysaccharides from the red seaweed Gracilaria dura (Gracilariales, Rhodophyta)

The yield and physical and chemical properties of agars from Gracilaria dura (C. Agardh) J. Agardh, harvested in Thau lagoon (Mediterranean sea, France), were investigated. The agar yield ranged from 32% to 35%. Gel strength of agar ranged from 263 to 600 g cm(-2), with the maximum observed in October. A positive correlation was found between agar yield and gel strength (r = 0.82; P < 0.01). The gelling temperature followed the same pattern of gel strength and also showed higher value in October (43 degrees C). The nitrogen content varied from 1.04+/-0.60% (June) to 4.70+/-0.01% (October). A positive correlation was noted between nitrogen content and gel strength (r = 0.77; P < 0.05). The 3,6-anhydrogalactose content ranged from 0.70 to 0.84 and showed monthly significant differences (P < 0.05). There was a positive correlation between 3,6-anhydrogalactose content and gel strength. The values of sulfate content were relatively constant during the studied period and no significant differences were observed. The relative high gel strength indicates that this species may be considered as source of agar for commercial use.     

Process optimization for the production of sugar for the bioethanol industry from sorghum,a non-conventional source of starch

A commercial preparation of -amylase, Biotempase, obtained from Biocon India Pvt. Ltd., and crude glucoamylase produced from Aspergillus sp. NA21 were used to hydrolyse sorghum powder, a non-conventional starchy substrate. Among various concentrations of starch (15–35%, dry weight/volume) tried for maximum liquefaction; slurry made with 25% substrate concentration proved optimal. An economical process of liquefaction was carried out using steam under pressure (0.2–0.3 bar, 104–105 °C) to liquefy a 25% slurry in just 45 min, contrary to a slower process carried out at 95 °C in a water bath. For liquefaction of starch a pH of 5.0 proved to be optimum. The dose of Biotempase as prescribed by the supplier could be reduced by 33% achieving the same degree of liquefaction, by addition of CaCl₂ to the starch slurry at the concentration of 200 mg/l. The conditions for the saccharification of liquefied starch were optimized to be 45 °C and pH 5.0, producing 90% saccharification in 24 h. Supplementation of divalent ions Ca²⁺, Mg²⁺ and Zn²⁺ in the process of saccharification showed no effect. Finally glucose was found to be the main hydrolysis product in the saccharification of sorghum starch.     

Seasonal changes in the chemical and gelling characteristics of agar from Gracilaria verrucosa collected in Turkey

The chemical and gelling properties of agar from G. verrucosa collected from Izmit bay in Turkey at different months of the year were studied. Purification of agar was performed by using amylase treatment and isopropyl alcohol precipitation. The phycocolloid content was between 24.0–43.0% of the algal dry weight and was maximum in summer collected algae. Relative total sulfate and 3,6-anhydrogalactose content in the agar were determined from the ratios of infrared spectroscopy band intensities at 1250/2920 cm^–1 and 930/2920 cm^–1, respectively. 3,6-anhydrogalactose and sulfate contents were the highest in agar from algae collected from June until November and January until July, respectively. The gel strength of native agar were the highest from in autumn collected algae and increased to about 1250 N m^–2 after alkali treatment. Thus, this study demonstrated that G. verrucosa from Turkey produces an agar with optimal chemical and gelling properties after alkali-treatment in fall and winter collected algae and may be used for industrial agar production.     

Comparison of the photosynthetic efficiency,agar yield,and properties of <Emphasis Type="Italic">Gracilaria salicornia</Emphasis> (Gracilariales,Rhodophyta) with and without adelphoparasite

The red seaweed Gracilaria salicornia is widely distributed along the Gulf of Thailand and Andaman sea coast. In nature, the adelphoparasite Gracilaria babae is mostly found to grow on this alga, and it appears as a gall with dark green or yellowish orange color. This study was conducted to examine the photosynthetic efficiency, agar yield, and properties of G. salicornia in plants with (PGs) and without the adelphoparasite (Gs). The results showed that the photosynthetic efficiency of the PGs and Gs was influenced by environmental factors. The agar yield extracted from the Gs was mostly lower than that from the PGs. The gelling temperature of agar solution from the PGs was lower than in the Gs but was not significantly different (p > 0.05). The gel melting point and the viscosity of the agar solution of the PGs was significantly higher than those of the Gs (p < 0.05). The gel strength and total carbohydrate content of the PGs agar were higher than those obtained from the Gs agar, except for the sample collected in June. The content of 3,6-anhydrogalactose was higher in the PGs than in the Gs collected in February and August, when the PGs agar had greater gel strength. Similarly, the PGs agar had a higher sulfate content than did the Gs agar. This study showed that changes in the photosynthetic efficiency, yield, and properties of agar extracted from G. salicornia were influenced by the environmental factors in association with the presence of the adelphoparasite G. babae.     

Production of fuel alcohol from oats by fermentation

Very high gravity (>30 g dissolved solids per 100 ml) mashes were prepared from hulled and hulless oats and fermented at 20° C with active dry yeast to produce ethanol. Excessive viscosity development during mashing was prevented by hydrolyzing -glucan with crude preparations of -glucanase or Biocellulase. Both these preparations possessed endo--glucanase activity. By using these enzymes and by decreasing the water to grain ratio, very high gravity mashes with low viscosity were prepared. Unlike wheat and barley mashes, oat mashes contained sufficient amounts of assimilable nitrogen to promote a fast rate of fermentation. The free amino nitrogen (FAN) content of oat mash could be predicted by the equation, mg FAN L^–1=8.9n wheren is the number of grams of dissolved solids in 100 ml of mash supernatant fluid. Ethanol yields of 353.2±3.7 L and 317.6±1.3 L were obtained per tonne (dry weight basis) of hulless (59.8% starch) and hulled (50.8% starch) oats respectively. The efficiency of conversion of starch to ethanol was the same in normal and very high gravity mashes.     

The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Effect of temperature on some characteristics of the thermostable α-amylase from Bacillus licheniformis

The V _max of an extracellular, thermostable -amylase from Bacillus licheniformis 44MB82 were 5.70×10^-3 and 9.70×10^-3 mM s^-1 at 30 and 90°C, respectively, whereas the K _m values were similar (0.9 mg ml^-1) at both temperatures. Excluding dextrins, the dominant products from soluble starch and amylopectin hydrolysis contained less than six glucose residues. The enzyme hydrolysed amylopectin better than soluble starch. Increasing the temperature from 30 to 90°C was accompanied by an increase in the production of malto-oligosaccharides, especially maltotetrose, and this was related to the secondary hydrolysis of maltopentose and maltohexose.The authors are with the Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113. 26 Academician G. Bonchev, Bulgaria     

Fast-germinating low β-glucan mutants induced in barley with improved malting quality and yield

Summary Mutation breeding has been used to improve the speed of germination in the high-yielding spring barley variety Troubadour. Five mutants were selected which combined fast germination and good agronomic performance. Two of these mutants yielded significantly more than did Troubadour over eight environments, and showed a clear improvement in their malting quality through an increase in extract yield. The improvement in malting quality appeared to be due to a decrease in the -glucan content, which seemed to enhance the germination speed and thus the starch degradation. The improvement in grain yield is postulated to be due to a better early growth caused by the enhanced germination speed. All the described changes could theoretically be explained by a single mutation event in each of the mutant genotypes, affecting the quantity of -glucans present in the endosperm.     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.     

Syntheses of hydroxybenzyl-α-glucosides by amyloglucosidase-catalyzed transglycosylation

Glycosides were synthesized from soluble starch and hydroxybenzylalcohols (o, m and p) by the transglycosylation activity of amyloglucosidase from Rhizopus sp. in a water system. The synthesized glycosides were identified as hydroxybenzyl--glucosides, of which one molecule of hydroxybenzylalcohol was bound to 1-OH of glucose. The transglycosylation reaction was performed in a reaction system containing 50 mg starch, 50 mg hydroxybenzylalcohol, and 10 units of enzyme in pH 5.0 at 45°C. The glycoside was rapidly hydrolyzed to glucose and hydroxybenzylalcohol by the -glucosidase.     

Purification and some properties of Α-amylase from an ectomycorrhizal fungus, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

-Amylase from a still culture filtrate of Tricholoma matsutake, an ectomycorrhizal fungus, was isolated and characterized. The enzyme was purified to a homogeneous preparation with Toyopearl-DEAE, gel filtration, and Mono Q column chromatography. The -amylase was highly purified (3580 fold) with a recovery of 10.5% and showed a single protein band by SDS-PAGE. The enzyme was most active at pH 5.0–6.0 toward soluble starch and stable within the broad pH range 4.0–10.0. This -amylase was a relatively thermostable enzyme (optimum temperature, 60°C; thermal stability, 50°C). The molecular mass was 34kDa by size-exclusion chromatography and 46kDa by SDS-PAGE. This enzyme was not inhibited by the Hg²⁺ ion. Measurement of viscosity and TLC and HPLC analysis of the hydrolysates obtained from amylose showed that the amylase from T. matsutake is an endo-type (-amylase). Substrate specificity was tested using amylose with different polysaccharides. This -amylase readily hydrolyzed the -1,4 glucoside bond in soluble starch and amylose A (MW, 2900), but did not hydrolyze the -1,6 bond and cyclic polysaccharides such as - and -cyclodextrin.     

High-Activity Barley \bold\ralpha-Amylase by Directed Evolution

Barley -amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations, Q44H and R303K, result in amino acids highly conserved in cereal -amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.