Production of panose from maltose by intact cells of Aureobasidium pullulans

Panose, a major component of isomalto-oligosaccharides, was selectively produced from maltose using transglucosylation reaction catalyzed by intact cells of Aureobasidium pullulans. When 50 %(w/v) maltose was used as a substrate, the maximum concentration of panose accumulated in the final reaction mixture was about 50 %(w/w) after 120 hr reaction at 55 °C.     

Rapid monitoring of autolysis process of proteases by capillary electrophoresis

A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 °C–50 °C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 °C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 °C. Similar results were obtained when MCP-01 was incubated at 20 °C, 40 °C and 50 °C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.     

Production of 1-kestose with intact mycelium of Aspergillus phoenicis containing sucrose-1F-fructosyltransferase

Summary Favourable reaction conditions for the enzymatic production of 1-kestose by sucrose-1^F-fructosyltransferase, SFT (EC 2.4.1.99) from Aspergillus phoenicis CBS 294.80 mycelium were established. The intracellular enzyme SFT works best at 60°C, exhibits a relatively high thermostability and possesses an alkaline pH optimum. An invertase also present in the mycelium of A. phoenicis possesses an acidic pH optimum. Consequently, around pH 8.0 sucrose is converted mainly to 1-kestose and nystose while fructose is only formed in relatively small amounts. Under optimal conditions (55° C, pH 8.0 and an initial sucrose concentration of 750 g 1^-1) a yield of about 300 g 1-kestose per 1.01 reaction mixture could be achieved after 8 h.Offprint requests to: J. A. M. van Balken     

Xylanase production in solid-state fermentation: a study of its properties

Summary Xylanase production by Aspergillus niger van Tieghem was studied in solid-state cultivation. The screening of substrates was carried out in column incubators aerated with humidified air at 30°C. Results of physiological studies showed that the best yield of xylanase was 2500 U/g dry matter on a mixture of straw+bran 1:1 at 70% of moisture content.In order to compare some properties of the xylanase produced in both liquid and solid cultures, A. niger was also grown on xylan in submerged cultures. The enzymes produced in solid and liquid cultures have an optimum pH of about 3.8 and 4.5, respectively. Xylanase synthetized in solid fermentation is a little more thermostable than that from liquid culture and is maximally active at 50° C, compared to 45° C for enzyme from liquid culture.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel

Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 °C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 °C. Similarly, at 50 °C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.     

Highly efficient production of monoglycerides by the continuous removal of fatty acids from lipase-catalyzed oil hydrolysis

Highly efficient production of monoglycerides was achieved from lipase-catalyzed oil hydrolysis by the continuous addition of CaCl₂ to remove the fatty acids produced. A fusion protein produced by connecting a cellulose-binding domain of Trichoderma hazianum cellulase to Bacillus stearothermophilus L1 lipase was used as a model 1,3-regiospecific lipase. The reaction was performed at pH 10 and 50°C, and the relationship between continuous removal of fatty acids and the production of monoglyceride was investigated by microscopic and HPLC analysis of oil emulsions and the reaction products. Without the addition of Ca^{2 +} the reaction was inhibited by fatty acids, with the decrease in reaction rate being proportional to the concentration of fatty acids. When CaCl₂ was continuously added in a 1:2 molar ratio with the released fatty acids, the reaction progressed unimpeded due to the formation of Ca-soaps. Both the yield and the fraction of monoglyceride in the reaction product increased due to the continuous removal of fatty acids.     

Sequential two-step conversion of 4-oxoisophorone to 4-hydroxy-2,2,6-trimethylcyclohexanone by thermophilic bacteria

Summary The sequential two-step conversion of 4-oxoisophorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone (4-HTMCH) via diyhdrooxoisophorone (DOIP) was achieved using two kinds of thermophiles, Thermomonospora curvata and Bacillus stearothermophilus. In the first step, 83% OIP was converted to DOIP by T. curvata during 12 h incubation at 50° C. The resulting reaction mixture containing thermophile cells, DOIP (2.5 mg/ml), and OIP (0.5 mg/ml) was used directly in the second step after adjusting to pH 7 and adding glycerol. In the second step, DOIP in the reaction mixture was converted to 4-HTMCH by B. stearothermophilus. The final concentration of 4-HTMCH and DOIP after 24 h of incubation was 2.5 mg/ml and 0.5 mg/ml respectively; OIP was not detected. The total conversion yield of 4-HTMCH from OIP was 83% through the two-step conversion. The two-step conversion by a sequential culture system using T. curvata and B. stearothermophilus was found to be suitable for 4-HTMCH production. Offprint requests to: I. Karube     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

Low temperature photo-oxidation of chloroperoxidase Compound II

Oxidation of the heme-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago with peroxynitrite (PN) gave the Compound II intermediate, which was photo-oxidized with 365 nm light to give a reactive oxidizing species. Cryo-solvents at pH ≈ 6 were employed, and reactions were conducted at temperatures as low as − 50 °C. The activity of CPO as evaluated by the chlorodimedone assay was unaltered by treatment with PN or by production of the oxidizing transient and subsequent reaction with styrene. EPR spectra at 77 K gave the amount of ferric protein at each stage in the reaction sequence. The PN oxidation step gave a 6:1 mixture of Compound II and ferric CPO, the photolysis step gave an approximate 1:1 mixture of active oxidant and ferric CPO, and the final mixture after reaction with excess styrene contained ferric CPO in 80% yield. In single turnover reactions at − 50 °C, styrene was oxidized to styrene oxide in high yield. Kinetic studies of styrene oxidation at − 50 °C displayed saturation kinetics with an equilibrium constant for formation of the complex of K_bind = 3.8 × 10⁴ M^− 1 and an oxidation rate constant of k_ox = 0.30 s^− 1. UV-Visible spectra of mixtures formed in the photo-oxidation sequence at ca. − 50 °C did not contain the signature Q-band absorbance at 690 nm ascribed to CPO Compound I prepared by chemical oxidation of the enzyme, indicating that different species were formed in the chemical oxidation and the photo-oxidation sequence.     

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus<Emphasis Type="Italic"> Sporotrichum thermophile</Emphasis>

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k _cat/K _m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl--l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.     

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl--d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.     

Résistance des abeilles (Apis mellifica L. var.ligustica) a des températures élevées

Summary The following is a study of resistance of worker honey bees (Apis mellifica L. var.ligustica) to high temperatures and of the effect of nutrition upon it.Survival of honey bees of spring generation was studied at 45, 50, 55 and 60°C during 15, 30, 45 and 60 minutes exposures. The survival rate was established at the end of their exposure, and 24 hours later. Lethal effects of heat were evident immediately after a 30 minute exposure to 50° C. These effects could be noticed in the survivers 24 hours following exposure.Effect of nutrition on heat resistance at 32° C and at 50° C was studied on one group of bees supplied with a 30 % honey solution; on another- with water only, and on a third group which served as a control (no food). Their survival at 32° C after 12 hours exposure was 100%, 81% and 48%, respectively. However, an exposure duration of 45 minutes at 50°C resulted in a survival of 22% of control bees as compared to a total survival of those fed on honey solution.     

Reaction of substituted phenols with thermostable laccase bound to Bacillus subtilis spores

A strain of B. subtilis produced 1.8 times more laccase on sporulation medium than on non-sporulation medium. Spores oxidized mono- and di-methoxyphenols (0.1 mM) at 50 °C. The half-life of laccase bound to spores was about 2 d and the substrate was repeatedly removed by spores recovered from the reaction mixture.     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Aspartame precursor synthesis in water miscible cosolvent catalysed by thermolysin

Summary The synthesis of the dipeptideN-benzyloxycarbonyl-L- aspartyl-phenylalanine methyl ester, aspartame precursor, catalysed by thermolysin in aqueous and aqueous methanolic solutions was studied. Thermolysin with concentration as low as 10 M in 25% methanol can catalyse the synthetic reaction. The optimum methanol compositions at 4°C and 37°C were 50% and 25% respectively where an increase in peptide yield of 85% was obtained for both conditions as compared to that in water.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-aspartic acid - L-Phe-OMe L-phenylalanine methyl ester - N-cbz-L-Asp-Phe-OMe N-benzyloxycarbonyl-L-aspartyl-phenylalanine methyl ester All the % of methanol is a volume % in water unless otherwise specified.     

Influence of freezing temperatures on a cactus,Coryphantha vivipara

Summary Coryphantha vivipara (Nutt.) Britton & Rose var. deserti (Engelm.) W.T. Marshall (Cactaceae) survived snow and tissue temperatures of-12°C in southern Nevada. However, the freezing point depression of the cell sap was only about 0.9°C. When the nocturnal air temperature in the laboratory was reduced from 10°C to-10°C for one night, the optimum temperature for CO₂ uptake shifted from 10°C to 6°C and uptake was reduced 70%, but full recovery to the original values occurred in 4 days. Nocturnal temperatures of-15°C killed 2 out of 5 plants and-20°C killed 5 out of 5, as judged by lack of net CO₂ uptake at night over a 2-month observation period. when the stems were cooled at 2° C/h, supercooling to about-6°C occurred followed by an exothermic reaction that presumably represented the freezing of extracellular water. When the subzero temperature was lowered further, no other exothermic reaction was observed and the cells became progressively dehydrated. Freezing-induced tissue death was ascribed to this cellular dehydration, which led to about 94% loss of intracellular water at-15°C. when the tissue temperature was lowered, the ability of chlorenchyma cells to plasmolyze and to take up a stain decreased, both being nearly 70% inhibited at-15°C and completely abolished at-20°C. Some cold-bardening occurred, since lowering the air temperature from 30° to-10°C in 10°C increments at weekly intervals caused the subzero temperature for 50% inhibition of staining to decrease from-10°C to-17°C. Extension of the range of C. vivipara to regions with wintertime freezing apparently reflects the tolerance of considerable freeze dehydration by its protoplasts.     

Survival of Coregonus albula (L.) (Teleostei) embryos incubated at different thermal conditions

Eggs of Coregonus albula were incubated at constant temperatures: 1.1, 2.0, 2.9, 4.9, 6.6, 8.4, and 9.9 °C, and the percentage of normal hatch was 20.6, 11.8, 30.4, 61.0, 51.7, 32.6, and 14.6%, respectively. The lower and upper median tolerance limit (TL 50) defined as the interpolated temperature at which embryos survival to hatch was 50% of the highest response (61% at 4.9 °C) were 2.9 and 8.5 °C, respectively. The optimum temperature range delimited by lower and upper TL 75 was encompassed by 4.0 and 7.2 °C.Eggs of C. albula incubated at variable temperature in a commercial hatchery showed a very high survival (up to 76%). Similarly low survival observed during hatching of embryos at constant temperatures of 1.1 and 2.0 °C could be hightened (to about 90%) by raising the temperature in the beginning of hatching period. This phenomenon was utilized in the technique of delaying C. albula embryos' mass hatching for the purpose of synchronization in time of stocking the lakes with the time of appearence of good thermal and food conditions for C. albula larvae.The conception of the optimal thermal conditions for Coregoninae embryogenesis was developed as the course of incubation temperature, securing the highest survival rate during embryogenesis and also during the larval period.     

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.