Antifungal Activity of Chitinases from <Emphasis Type="Italic">Trichoderma aureoviride</Emphasis> DY-59 and <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> VS-9

Two chitinolytic fungal strains, Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil samples of Korea and Vietnam, respectively. DY-59 and VS-9 crude chitinases secreted by these fungi in the 0.5% swollen chitin culture medium had an optimal pH of 4 and the optimal temperatures of 40°C and 60°C, respectively. Enzymatic hydrolysis products from crab swollen chitin were N-acetyl-β-D-glucosamine (GlcNAc) by DY-59 chitinase, and GlcNAc and N, N′-diacetylchitobiose (GlcNAc)₂ by VS-9 chitinases. The chitinases degraded the cell wall of Fusarium solani hyphae to produce oligosaccharides, among which GlcNAc, (GlcNAc)₂, and pentamer (GlcNAc)₅ were identified by high-pressure liquid chromatography. DY-59 and VS-9 chitinases inhibited F. solani microconidial germination by more than 70% and 60% at final protein concentrations of 5 and 27 μg mL⁻¹, respectively, at 30°C for 20 h treatment.     

Temperature-dependent development and life table parameters of <Emphasis Type="Italic">Typhlodromus bagdasarjani</Emphasis> (Phytoseiidae) fed on two-spotted spider mite

The predatory mite Typhlodromus bagdasarjani Wainstein and Arutunjan (Acari: Phytoseiidae) is an indigenous and widespread species of the Middle East fauna. In this paper we assess the effect of temperature on developmental rate and reproduction potential of T. bagdasarjani under laboratory conditions. The development of this species was determined at 15, 20, 25, 30, 35 and 37.5 ± 1°C, 60 ± 10% RH and L16:D8 h photoperiod. The total developmental time averaged 28.2, 15.0, 8.9, 7.6, 7.2 and 7.4 days at 15–37.5°C, respectively, when feeding on immature stages of two-spotted spider mite, Tetranychus urticae Koch. The lower developmental threshold (T ₀ ) and thermal constant (K) for the development of this predator were estimated 9.2°C and 162 degree-days by the Ikemoto linear model. The life table parameters were estimated at 15–35°C. The shortest life span of females at 35°C was 45.0 days, followed by 50.7, 50.9, 103.3 and 136.8 days at 30, 25, 20 and 15°C, respectively. Mated females laid on average 19.9, 26.3, 41.1, 39.6 and 31.3 eggs per female at 15–35°C, respectively. The intrinsic rate of increase (r _m ) and finite rate of increase (λ) increased significantly with increasing temperature. The r _m values ranged from 0.021 (15°C) to 0.186 (35°C) days⁻¹. The highest value of net reproductive rate (R ₀) was 13.6 females progeny/female/generation at 25°C. The results demonstrated that T. bagdasarjani is well adapted to high temperatures. However, the efficiency to control spider mites may be affected by behavioral characteristics of the predator and its prey under real conditions.     

The Endopolysaccharide Metabolism of the Hyperthermophilic Archeon <Emphasis Type="Italic">Thermococcus hydrothermalis</Emphasis>: Polymer Structure and Biosynthesis

The endopolysaccharide accumulated by Thermococcus hydrothermalis was extracted and purified from a 4 h culture. It presented an “amylopectin-like” structure with an average chain length of 14 and a ramification degree of 7.5%. The glucosyltransferase was isolated, partially purified and characterized. The molecular mass was 42 kDa by SDS PAGE and 85 ± 5 kDa by gel filtration. This enzyme was able to use both Uridine-5′-DiPhosphoGlucose (UDPG) and Adenosine-5′-DiPhosphoGlucose (ADPG) as substrates. Optimal pH and temperature for the enzyme were 5.5 and 80°C, respectively. In the presence of 3.2 mM ADPG, the half life of the protein was 6 min at 110°C. The apparent K _m value with the two substrates was 0.9 mM, but the V _max was 9.7 fold higher for ADPG. A branching activity was also detected at high temperature, up to 80°C by different methods: phosphorylase stimulation, iodine, and branching linkage assays.     

Cloning and bioinformatics analysis of an endoglucanase gene (Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris

Using 3′ and 5′ rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A, named as Aucel12A, is 1,027 bp in length harboring 5′ and 3′ non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence is 1,576 bp in length, consisting of a 5′ flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp. The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W. of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5–7.0, and at a temperature of 55°C or below. Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag⁺, Hg²⁺ and Fe²⁺. The K _m and V _max of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively.     

Specialty Fats from Beef Tallow and Canola Oil: Establishment of Reaction Conditions, Characterization of Products, and Evaluation of Crystal Stability

The effect of reaction time (0–90 min), catalyst concentration (0.2–0.5% CH₃ONa powder), and temperature (60–90 °C) of chemical interesterification (CIE) was studied to determine the optimal conditions for maximal change of solid fat content (SFC) in minimal time in a beef tallow (BT)/canola oil (CaO) blend (80:20, w/w, herein after referred to as BT/CaO blend 80:20). The optimal conditions were obtained as: CH₃ONa 0.4%, 60 °C, 30 min. BT/CaO blends (60:40, 65:35, 70:30, 75:25, 80:20, and 85:15) were each interesterified on a laboratory scale under afore-determined optimal conditions, and the corresponding 12 samples, before and after CIE, were characterized in terms of the SFC profile and compatibility. SFC profiling showed an increase in SFC (<5%) between 5 and 21.1 °C and a slight drop (<3%) in SFC between 40 and 45 °C for the interesterified blends. Compatibility analysis showed the presence of monotectic systems in original blends, proven through isothermal solid diagrams and isosolid diagrams. The incompatibility among the fats and oils was eliminated after reaction and the solution behavior shifted to a continuous solid solution. The interesterified 85:15, 80:20/75:25, 70:30, 65:35, and 60:40 BT/CaO blends displayed characteristics suited to application, respectively, for bakery shortenings, frying fats, all-purpose bakery shortenings, and bakery and roll-in margarines. The model shortenings produced from interesterified blends had more stable crystal morphology, crystal sizes, and double-chain (2L) stacking β′ polymorphs than blended shortenings under temperature fluctuation storage. Sensory analysis also showed that the former had less graininess and better spreadability than the latter during storage.     

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from <Emphasis Type="Italic">Staphylococcus</Emphasis> sp. strain AJ isolated from Korean salt-fermented Anchovy-<Emphasis Type="Italic">joet</Emphasis>

A novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5–6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5–3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K _m) and K _cat values of AJ towards α-casein were 0.38 mM and 19.73 s⁻¹, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.     

Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD⁺ or NADP⁺ as the coenzyme but preferred NAD⁺. The K _m values for malic acid, NAD⁺ and NADP⁺ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.     

Cross-mating experiments with geographically different populations of <Emphasis Type="Italic">Amblyomma cajennense</Emphasis> (Acari: Ixodidae)

The effect of temperature on the development and fecundity of Sancassania polyphyllae fed on tissues of Polyphylla fullo larvae was studied at 15, 20, 25, 30, and 35 ± 1°C and 65 ± 10% RH in a dark incubator. Mean developmental period of immature stages decreased significantly with increasing temperatures from 15 to 30°C. Developmental periods at 30–35°C were not significantly different. The estimated lower developmental thresholds of the various immature stages ranged between 10.1 and 11.5°C. The thermal constant for the egg-to-female adult was 93.5 degree-days. The pre-oviposition, oviposition, and post-oviposition periods and female longevity were significantly longer at 15°C than at higher temperatures. Mean total and daily fecundity were the highest at 25°C, which were significantly different from those obtained at 15, 20 and 30°C. The net reproductive rate (R ₀) was the highest at 25°C (588.3 ♀/♀). The longest mean generation time (T ₀) occurred at 15°C (36 days) and the shortest occurred at 30°C (9.2 days). The highest intrinsic rate of increase (r _m) for S. polyphyllae was observed at 25 (0.61 ♀/♀/day) and 30°C (0.62 ♀/♀/day).     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

Temperature-dependent development and reproductive traits of <Emphasis Type="Italic">Tetranychus macfarlanei</Emphasis> (Acari: Tetranychidae)

Development and reproductive traits of Tetranychus macfarlanei Baker & Pritchard (Acari: Tetranychidae) were investigated on kidney bean, Phaseolus vulgaris L., at eleven constant temperatures. Tetranychus macfarlanei was able to develop and complete its life cycle at temperatures ranging from 17.5 to 37.5°C. At 15 and 40°C, a few eggs (2–4%) hatched but further development was arrested. Development from egg to adult was slowest at 17.5°C and fastest at 35°C for both females and males. Using Ikemoto and Takai’s linear model, the estimated lower developmental thresholds for egg-to-female adult, egg-to-male adult and egg-to-egg development were 12.9–13.0°C. The thermal constants for the respective stages were 110.85, 115.99 and 125.32 degree-days (DD). The intrinsic optimum temperatures (T _Φ) calculated by non-linear SSI model were determined as 24.4, 24.4 and 24.2°C for egg-to-female adult, egg-to-male adult and egg-to-egg development, respectively. The net reproductive rate (R ₀) was highest at 25°C (167.4 females per female) and lowest at 17.5°C (42.6 females per female). The intrinsic rate of natural increase, r _m, increased linearly with the rising of temperature from 0.102 at 17.5°C to 0.441 day⁻¹ at 35°C. These values suggested that T. macfarlanei could be growing quickly in response to increasing temperatures from 17.5 to 35°C and provide a basis for predicting its potential geographical range.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

Simultaneous production and characterization of medium-chain-length polyhydroxyalkanoates and alginate oligosaccharides by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> NK-01

When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L⁻¹ medium-chain-length polyhydroxyalkanoate (PHA_MCL) and 0.57 g L⁻¹ alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHA_MCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T _m and T _g values for the PHA_MCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was 700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3.     

Stabilization of native and double <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidases from <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> and <Emphasis Type="Italic">Trigonopsis variabilis</Emphasis> by immobilization on streptavidin-coated magnetic beads

Double d-amino acid oxidases (dRtDAO and dTvDAO) were previously genetically constructed by linking the C-terminus of one subunit of their corresponding native DAOs from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) to the N-terminus of the other identical subunit. We have now immobilized these double DAOs and their native counterparts onto streptavidin-coated magnetic beads through the interaction between biotin and streptavidin. The catalytic efficiencies (k_cat/K_M) of immobilized DAOs toward d-alanine and cepharosporin C remained similar to those of their soluble forms, except the catalytic efficiency of immobilized TvDAO toward d-alanine was decreased by 56%. After immobilization, the T_m value for RtDAO was shifted 15°C higher to 60°C, while those for dRtDAO, TvDAO and dTvDAO were increased by 5–8°C to 56, 60 and 60°C, respectively. In the presence of 10 mM H₂O₂, immobilized RtDAO, dRtDAO, TvDAO and dTvDAO exhibited half-lives of about 8, 10, 3 and 5 h, respectively, giving 16-, 10-, 6- and 7-fold greater stability than their soluble forms, respectively. Therefore, immobilization through biotin–streptavidin affinity binding enhances the thermal and oxidative stability of native and double DAOs studied, especially RtDAO. The additive stabilizing effect of subunit fusion and immobilization was more pronounced in the case of RtDAO than TvDAO.     

Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite

Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after incubation for 3 h. The K _m and V _max values of the immobilized enzyme were found to be 0.0619 mmol l⁻¹ and 0.147 mmol h⁻¹ mg⁻¹, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles of reuse at 37 °C.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Substrate specificity characterization of a thermostable keratinase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> KS-1

A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential β-keratin-degrading enzyme with prospective applications in degradation of β-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS–PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60°C, respectively. The enzyme was highly thermostable with a t _1/2 > 2 h at 80°C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val¹²-Glu¹³, Ala¹⁴-Leu¹⁵, Gly²⁰-Glu²¹ and Arg²²-Gly²³ residues.     

Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication