Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

Kinetic and thermal stability studies of rulactine,a proteolytic enzyme fromMicrococcus caseolyticus

Summary Kinetics and thermostability of Rulactine, a protease fromMicrococcus caseolyticus were investigated. Study of the enzyme activity as a function of the temperature showed an optimum peak of 45°C. The effeci of the substrate concentration on the initial velocity at various temperatures was examined, and V_max and K_M were determined using a Lineweaver-Burk reciprocal plot. The activation energy evaluation gave a value of 9500 cal/mole. Studies of additives such as polyhydric alcohols (glycerol, erythritol, xylitol and sorbitol) and disaccharides (sucrose and lactose) to Rulactine at 58°C proved that they have a stabilizing effect on Rulactine.     

Effect of glycerol,polyethylene glycol and glutaraldehyde on stability of phenylalanine ammonia-lyase activity in yeast

Summary The polyhydric alcohols, glycerol and sorbitol, significantly increased the rate ofl-phenylalanine production from trans-cinnamic acid using whole cells ofRhodotorula rubra. Chloride ions and oxygen prevented the stimulatory effect of the polyhydric alcohols. Furthermore, the severe inhibition, of the biotransformation by high trans-cinnamic acid concentrations was alleviated in the presence of glycerol, and sorbitol. The rate of conversion could be manipulated still further, even with high trnas-cinnamic acid concentrations, by elevating the reaction pH to, 12 in the presence of polyhydric alcohol. When cells were also treated first with glutaradehyde (0.1% v/v) and then polyethylene glycol (15% v/v), although neither compound stimulated the actual rate of bioconversion, the reaction was markedly stabilised and gave a 73% yield after 28 days of continuous operation.     

Purification and characterization of 4-N-trimethylamino-1-butanol dehydrogenase from Fusarium merismoides var. acetilereum

From investigation of 60 filamentous fungi, we identified Fusarium merismoides var. acetilereum, which uses 4-N-trimethylamino-1-butanol (TMA-butanol) as the sole source of carbon and nitrogen. The fungus produced NAD⁺-dependent TMA-butanol dehydrogenase (DH) when it was cultivated in medium containing TMA-butanol. The enzyme showed molecular mass of 40 kDa by SDS–PAGE and 160 kDa by gel filtration, suggesting that it is a homotetramer. TMA-butanol DH is stable at pH 7.5–9.0. It exhibits moderate stability with respect to temperature (up to 30 °C). Additionally, it has optimum activity at 45 °C and at pH 9.5. The enzyme has broad specificity to various alkyl alcohols and amino alkyl alcohols, and the carbon chains of which are longer than butanol. Moreover, the activity is strongly inhibited by oxidizing agents, carbonyl and thiol modulators, and chelating agents. This report is the first study examining TMA-butanol DH from eukaryotic microbes.     

Physical and chemical changes associated with seasonal alterations in freezing tolerance in the adult northern tenebrionid, Upis ceramboides

The adult tenebrionid beetle Upis ceramboides overwinters in the northern taiga forests of North America in a hibernaculum typically just beneath loose tree bark above the snowline. The beetles may be exposed to temperatures as low as ?55°C, which is approximately the lower limit of cold tolerance found in specimens collected in mid-winter. Supercooling points average ?6.3°C throughout the year and, contrary to expectation, show no seasonal variation in spite of major alterations in haemolymph composition and freezing tolerance. Summer beetles are incapable of withstanding temperatures below the supercooling point but freezing tolerance increases during the fall (September–November) and the lower lethal temperature (LLT) is maintained at ca. ?55°C until March, after which it gradually rises to the summer level of ?6°C. Changes in freezing tolerance are closely associated with seasonal alterations in the polyhydric alcohols sorbitol and threitol. Neither polyol is present in measureable amount during summer; sorbitol accumulates to an average haemolymph concentration of 0.44 M/l in winter and threitol reaches 0.25 M/l. Summer beetles contain about 14% more water than beetles collected during the other seasons. Upis ceramboides thus undergoes unique seasonal changes in physical and chemical characteristics that enable it to tolerate severe, prolonged subfreezing temperatures.     

Effect of polyols on the thermostability of pullulan-hydrolysing activity from Sclerotium rolfsii

Summary The influence of various polyols on the thermostability of pullulan-hydrolysing activity fromSclerotium rolfsii was studied. The half-life of the enzyme activity at 60°C was determined to be of the order of 30 min. In the presence of xylitol and sorbitol (3 M or more) there was a significant enhancement in the thermostability of the enzyme with retention of 100% activity after incubation for 7 h at 60°C. However, ethylene glycol and glycerol were found to have no protective effect. The stabilizing efficiency was found to be dependent on the concentration of the polyhydric alcohol used and the number of OH-groups present per molecule.     

Influence of glycerol and other polyhydric alcohols on the quaternary structure of an oligomeric protein

Dissociation of tetrameric l-asparaginase from Escherichia coli B was examined in the presence of urea containing one of the following polyhydric alcohols: ethylene glycol, 1,2-propanediol, 1,3-propanediol, glycerol, erythritol, arabitol, adonitol, mannitol, sorbitol, inositol, glucose, sucrose, and polyethylene glycol. Low concentrations of these compounds accelerate the rate of subunit dissociation, and, with the exception of the propanediols and polyethylene glycol, higher concentrations decrease the rate at which the oligomeric enzyme dissociates. The specific concentration at which this transition occurs is related to the length of the carbon chain of the polyhydric alcohols and to the steric configuration of the hydroxyl groups about the asymmetric carbon atoms. In addition, the rate at which the oligomeric enzyme dissociates decreases as the number of hydroxymethyl groups per molecule polyol increases and reaches a maximum with the six carbon members.Low concentrations (1% by volume) of methanol, ethanol, ethylene glycol, propylene glycol, or glycerol contained in the renaturation buffer slightly accelerate the rate of reassembly of denatured subunits. The rate at which reassociation to the tetramer occurs also increases as the number of hydroxymethyl groups per molecule of polyhydric alcohol increases.     

Characterisitics and Gene Cloning of Phospholipase D of the Psychrophile,Shewanella sp.

Phospholipase D, with a molecular mass of 64 kDa, was purified from the psychrophile, Shewanella sp. The enzyme showed maximal activity at pH 7.8 and 40 °C in the presence of the Ca²⁺-ion, and its activity at 10 °C was 6.5% of maximum. The enzyme exhibited high activity to the non-micelle form of phosphatidylcholine in an aqueous solution containing water miscible alcohols such as methanol, ethanol, iso-propanol, and n-propanol. Nucleotide sequencing of the enzyme gene yielded a deduced amino acid sequence, which showed 36.2% identity to that of Streptomyces chromofuscus phopsholipase D alone. The low sequence similarity to other phopsholipase D enzymes suggests that the purified enzyme might be a novel phospholipase D.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N,?N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1?M solute were heated for 10?min at temperatures between 35–75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1?M stabilizer solutions were frozen at ?30°C and freeze-dried for 24?h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze‐induced lesions: expansion‐induced lysis (EIL) between ?2 and ?4 °C and loss of osmotic responsiveness (LOR) between ?5 and ?12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at ?5 and ?6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at ?5 and ?6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low‐temperature‐induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.     

Highly stable laccase from repeated-batch culture of Funalia trogii ATCC 200800

The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN₃ and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.