A cellulase-free xylanase from alkali-tolerantAspergillus fischeri Fxn1

Summary An alkali-tolerant fungusAsperqillus fischeri Fxn1 isolated from xylan enrichment grew in the pH range 5–10 and secreted an extracellular cellulase-free xylanase. Arabinose, lactose, maltose, cellobiose and glucose induced low levels of xylanase (1.8–9.0 IU/ml), whereas xylose, xylan and wheat bran induced higher level (34–45 IU/ml).CMcellulose and FPcellulose did not support growth. The optimum pH of xylanase was 6.0–6.5 and it was stable in a wide range of pH 5–9.5. The optimum temperature was 60°C and it was stable upto 55°C. The half-lives at 50 and 55 °C were 240 and 40 min. respectively. This enzyme released reducing sugars from pulp at pH 9.0 and 40°C.     

Properties of extracellular proteases from three psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil

Three Antarctic psychrotolerant Stenotrophomonas maltophilia were isolated and the characteristics of their extracellular serine proteases were described. The isolates were able to grow at 14 and 34°C, but grew better between 20 and 28°C. The highest protease secretion was reached at 20–24°C. The purified enzyme preparations had maximal activity at 55–60°C and alkaline pH. They showed high pH stability, retaining more than 60% of residual activity after 3 h of incubation at a pH range of 4–12. The thermal stability was slightly lower compared with a commercial mesophilic protease, with 74–79% residual activity after 90 min at 40°C and 50% inactivation at 50°C between 43 and 69 min. These properties suggest that the Antarctic isolates could be adapted to cold by means of synthesising more enzymes with high activity but that the proteases they produce are not truly cold-active, being more similar to mesophilic enzymes.     

Microbiological process for the removal of Cr(VI) from chromate-bearing cooling tower effluent

Summary A microbiological process using Pseudomonas mendocina was developed for the removal of Cr(VI) from cooling tower effluent. The process, when carried out in a 20 liter continuous stirred tank reactor removed 25–100 mg chromate/l in 4.5–8 h with >99.9% efficiency in the presence of sugarcane molasses as nutrient. The process could sustain wide variations in pH (6.5–9.5), temperature (25°C–40°C) and was unaffected by commonly used biocides.     

Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay,southern Japan

Plants of Gracilaria sp.(chorda type), which grow along the coast of Uranouchi Inlet in Tosa Bay, southern Japan, showed the highest biomass in the summer (26 °C to 31 °C) and spring season (15.1 °C to 24.9 °C). Maximum biomass was 6952 g m^–2 in July, but gradually decreased in the autumn (30.5 °C in September to 20 °C in November) and winter (19.5 °C in December to 14.9 °C in February). Variation in yields and gel strength of the agars, were shown to depend on the time in the season. After alkali treatment (5% NaOH, 2 h) at three different temperatures (70, 80, and 90 °C), the agars showed gel strengths essentially that of commercial grade agars, with the best gel obtained at 80 °C. Maximum gel strength (1455 g cm^–2 of 1.5% agar gel) occurred in winter when the biomass and agar yield were low. Minimum gel strength was in spring. Gel strength was inversely correlated with agar yield, but was positively correlated with apparent viscosity. Maximum viscosity was 40 cP. in December. Gelling temperatures, pH of 1.5% agar gel, and moisture content in agars showed little variation.     

Elution of Trichoderma reesei cellulase from cellulose by pH adjustment with sodium hydroxide

Summary Up to 45% of bound cellulase (Avicelase) could be recovered from crystalline cellulose by pH adjustment with NaOH (0.008–0.01 M) to pH 10.0 (40°C). Enzyme was simultaneously desorbed and inactivated as the pH increased and both events occurred within 1 minute. Enzyme desorption and stability were also temperature dependent and desorption increased to approximately 65% by adding detergents.     

Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin

Summary An extracellular neutral protease, of Bacillus stearothermophilus KP 1236 (a soil isolate) able to grow at 39°–71°C was purified to homogeneity. The molecular weight, sedimentation coefficient in water at 20°C, and isoelectric point were estimated as 33,000, 3.46 S and 7.5, respectively. The enzyme was most active at 80°C and pH 7.0. The activity was stable for 10 min up to 80°C at pH 7.5 and for 18 h at 60°C over pH 6.0–8.8. The enzyme and thermolysin (microbial metalloproteinase, EC 3.4.24.4) shared their antigenic determinants in part.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan on Sapporo, July 30, 1985 (Abstracts, p. 333)     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Partial characterization ofAspergillus fumigatus polygalacturonases for the degumming of natural fibers

Summary Aspergillus fumigatus strain 4, cultured on citrus pectin as the sole carbon source, produced polygalacturonases whose activity was optimum at 65°C and pH 3.5–4.5. The enzymes presented a bimodal thermostability for 10 min, but not 60 min, of incubation. Polygalacturonases showed pH stability between 3.0 to 9.0. The enzymes were stable when stored at 4–6°C for 90 days, but their activity was reduced by 24% when they were stored at 26–30°C. Orange pulp was the best pectic carbon source tested for the production of pectinases capable of retting ramie fibers. The reutilization of these enzymes was possible, suggesting the viability of industrial use of pectinases for degumming ramie fibers.     

Critical analysis of factors influencing sphaeroplast generation from Saccharomyces cerevisiae

Efficient synthesis of large numbers of viable sphaeroplast from Saccharomyces cerevisiae has been found to be influenced by a number of factors. In this case, Trichoderma harzianum, NCIM 1185, culture filtrate has been used to prepare sphaeroplast from Saccharomyces cerevisiae, NCIM 3288. A method has been devised to isolate large number of viable sphaeroplast from the cell. Detailed analysis of various factors affecting the formation of sphaeroplasts from Saccharomyces cerevisiae has not yet been reported. This study showed critical analysis of various factors which influenced sphaeroplast formation. Most suitable conditions were: Age of the organism in slant — 1 d, cell age in liquid medium — 24 h, time of incubation of cell with 0.3% -mercaptoethanol — 30 min, level of lytic ezyme concentration — 79.2 ml, concentration of cell (dry wt. equivalent) — 0.1262 g, time of contact with lytic enzyme — 25 min, temperature of sphaeroplast formation — 30 °C, phosphate buffer — 25 mM of pH 6.5 and KCl as osmotic stabilizer — 0.7 M.     

Vorläufige Ergebnisse von Untersuchungen an eingefrorenen und bei tiefen Temperaturen bestrahlten Lymphozyten

Zusammenfassung Lymphozytenkulturen werden nach Zugabe von Glyzerin als Gefrierschutzsubstanz zunächst gemeinsam mit ungefähr 1 °C/min Abkühlgeschwindigkeit bis zum vollständigen Erstarren eingefroren und dann mit verschiedener Geschwindigkeit ( 200 °C pro min und 3 bis 5 °C/min) auf verschieden tiefe Temperaturen (–22, –80 und –196 °C) gebracht. Alle eingefrorenen Proben weisen nach 24 h Kulturdauer eine kleinere, nach 48 h Kulturdauer eine wesentlich höhere Mitoserate als die Kontrollgruppe (unbehandelte, nicht eingefrorene Plasmaproben) auf. In den schnell auf –196 °C abgekühlten Proben wurden in beiden Fällen (24 h und 48 h Kulturdauer) keine Metaphaseplatten gefunden. Die Zellkonzentrationen waren nur bei den schnell auf –196 °C abgekühlten Kulturen stark verringert. Die Chromosomenaberrationsrate der eingefrorenen Kulturen ist nicht signifikant erhöht.

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Preliminary results of investigations of lymphocytes, frozen and irradiated at low temperaturesI. The effects of cooling rate, low temperature, and incubation time on frozen lymphocytes

Summary Peripheral human lymphocytes were cooled to the temperature of solidification at a rate of less than 1 °C/min using glycerol as a protective agent against the effects of freezing. After solidification at temperatures of –10 to –15 °C and cooling to about –22 °C one group of the samples was thawn and the others were cooled rapidly to –80 or –196 °C by immersion into solid carbon dioxide or liquid nitrogen. Other samples of the frozen cell suspension were cooled down to the same temperatures much slower at a cooling rate of 3 to 5 °C/min. After rapid thawing in a 25 °C water bath the cell suspensions were removed from glycerol and cultured for 24 or 48 h before stopping mitoses by adding colchicine. The samples frozen at –22 °C, the samples cooled both rapidly and slowly to –79 °C, and the ones cooled slowly to –196 °C showed a lower rate of mitosis when colchicine was added after 24 h and a significantly higher rate of mitosis after 48 h of incubation before adding colchicine as compared to the controls (untreated, unfrozen plasma). In the culture frozen rapidly to –196 °C no metaphases could be found. The cell concentrations before and after freezing showed no significant differences except those of the culture frozen rapidly to –196 °C. The chromosome aberration rate is not significantly increased.

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Herrn Prof. Dr. H. A. Künkel zum 60. Geburtstag gewidmet.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Glucose oxidase-rich Aspergillus niger strain,cost-effective protocol and an economical substrate for the preparation of tablet grade calcium gluconate

Summary An optimized 25 litre scale protocol for the submerged batchwise preparation and recovery of calcium gluconate is devised. The optimal parameters of production envisaged the use of (a) glucose oxidase-rich Aspergillus niger strain, (b) salts-fortified high DE starch hydrolysate as the production medium, (c) pH 6.5 ± 0.1, 29 ± 1°C, 250 ± 10 rpm, (d) 1.0–1.5 vvm rate of aeration over 24 ± 2 h duration and (e) intermittant neutralization with calcium carbonate. The recovery protocol comprised of (a) charcoal decolourisation (2%, 60°C, l h), (b) filtration and methanol-aided precipitation, (c) centrifugation (700 g, 27°C), (d) vacuum drying (700 mm of Hg, 45°C) and (e) pulverization to provide 80% recovery of calcium gluconate, passing Indian/British pharmacopoeial specifications for the tablet grade preparation.     

Production Dynamics and Characterization of Chitinolytic System of Streptomyces sp. NK1057, a Well Equipped Chitin Degrader

Streptomyces sp. NK 1057 produced four extracellular chitinases. Two (62 and 48 kDa) were endochitinaseswhile the other two (35 and 28 kDa) had chitobiosidaseactivity. Chi62 was produced in early growth phase whereas the other three were produced later. Chi48 was optimally active at pH 4.0 and 60 °C whereas Chi35 was optimally active at pH 6.0 and 40 °C. Both the enzymes had fairly good pH and temperature stability up to 50 °C, with Chi48 being more thermostable than Chi35. Chi48 was significantly inhibited by N-acetylglucosaminebut not by Hg²⁺ and the reverse was true for Chi35. Chi48 and Chi35 had isoelectric points of 5.1 and 6.0 and the N-terminal amino acid sequence of Chi35 determined uptofour amino acids, was Gln–Ser–Pro– Gly. Chi62 and Chi48 were able to inhibit the germination of Fusariumoxysporum spores by 57.4 and 61.2% respectively whereas; Chi35 and Chi28 did so by only 14.1 and 3.8%, suggesting endochitinaseto possess antifungal activity. Only Chi28 exhibited a lytic activity, 0.022 U ml^–1, towards Micrococcus lysodeikticus cells.     

Transglycosylation of Stevioside by Cyclodextrin Glucanotransferases of Various Groups of Microorganisms

Cyclodextrin glucanotransferases (CGTases, EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside (in order to remove bitterness and aftertaste), with cyclodextrins (CDs) being used as donors. It was shown that CGTases produced by extremophilic microorganisms are effective biocatalysts. Optimum temperature and pH of these enzymes were 45°C and pH 6.5–7.5, respectively. The optimum stevioside-to-CD ratio and total concentration of dry matter for the synthesis of the best-tasting product were 1 : 1 (w/w) and 11.6%, respectively.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

Acylation of sucrose with vinyl esters using immobilized hydrolases: demonstration that chemical catalysis may interfere with enzymatic catalysis

The acylation of sucrose with vinyl laurate in dimethylsulfoxide was catalyzed by Celite (28% conversion in 24 h at 40 °C, 150 mg catalyst ml^–1), Eupergit C (11% conversion in 24 h at 60 °C, 150 mg catalyst/ml), and even the simple Na₂HPO₄ (17% conversion in 24 h at 40 °C, 20 mg catalyst ml^–1). These chemical acylations must therefore be taken into account in acylations of hydroxyl-containing compounds with enol esters in polar solvents using immobilized enzymes.     

Xylanolytic activities ofSpirochaeta thermophila

Spirochetes capable of degrading xylan or cellulose have not been commonly isolated, nor have their polysaccharolytic activities been characterized.Spirochaeta thermophila strain RI 19.B1 is xylanolytic and grows well at 65°C with oatspelt (OX), birchwood (BX), corncob (CCX-A) xylans, or glucuronoxylan (MGX) as the energy source. All xylans were extensively degraded and utilized during growth. About 72–82% of the initial hexuronic acids and 57–79% of initial pentoses disappeared during growth.S. thermophila possessed xylanase, xylosidase, and arabinofuranosidase enzyme activities. Low levels of these activities were detected with growth on glucose, but high expression of these activities occurred during growth on OX. All three activities were cell-associated and were more stable in cells than cell extracts. Xylan-degrading activities were measured with cells or cell extracts exposed (60 min) to a variety of temperatures (65°–85°C) and pHs (5.0–8.0). More than 50% loss of activities occurred at temperatures above 75°C. Although pH stability was affected by buffer, the optimal range was pH 6.5–7.5. These temperature and pH profiles for xylan-degrading activities coincide with those found for the growth ofS. thermophila.     

Isolation and partial characterization of bacteriocins from<Emphasis Type="Italic"> Pediococcus</Emphasis> species

Lactic acid bacteria have received increased attention as a potential food preservative due to their strong antagonistic activity against many food-spoilage and pathogenic organisms. Three Pediococcus species, P. acidilactici NCIM 2292, P. pentosaceous. NCIM 2296 and P. cervisiae NCIM 2171, were evaluated for bacteriocin production. Inhibitory substance were produced during the late growth phase and maximum production occurred at 37 °C after 36–48 h of incubation. Bacteriocins partially purified from these species by cold-acetone precipitation at 0 °C and cell adsorption desorption techniques have a broad inhibitory spectrum against microorganisms, including gram-negative bacteria such as Escherichia coli and Pseudomonas. Proteolytic enzymes inactivated these peptides, but amylase and lipase did not show any effect. The bacteriocins were stable over a wide pH range (3–8) and apparently most active at pH 4.0–5.0. They were heat-stable (1 h at ~80 °C and autoclaving) at pH 5.0. No loss in activity was observed when stored under refrigeration (4–8 °C). Tris-Tricine SDS-PAGE revealed the molecular masses of these peptides to be between 3.5 and 5.0 kDa.