Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures

A large increase in the activity of an enzyme involved in chlorogenic acid metabolism, hydroxycinnamyltransferase occurs in tomatoes stored at low temperatures. In contrast, the activity of the enzyme remains constant or falls slightly during normal ripening at 20°. The rise in activity occurs at temperatures below 10° and fails to occur at 15° or 20°. This increase in activity during low temperature storage occurs with fruit at all stages of ripening from mature green to fully ripe. The hydroxycinnamyltransferase of chilled tomatoes falls rapidly on transfer to 20° with a lag of about 4–8 hr and within 48 hr returns to that of unchilled fruit. The effects of such warming treatments are reversible since when a chilling period is resumed following warming to 20°, the rise in hydroxycinnamyltransferase activity is also resumed. Of the 5 other enzymes of phenylpropanoid metabolism studied, only PAL shows a similar increase in activity during low temperature storage although the activity of the other enzymes was maintained at higher levels in fruit at 2° than at 20°. The possible relationship between the behaviour of hydroxycinnamyltransferase activity at various temperatures and the known susceptibility of tomatoes to chilling injury is discussed.     

Studies on Tannin Acyl Hydrolase of Microorganisms

Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.

The molecular weight of the enzyme estimated by gel filtration method was about 200,000.

The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.

Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water.     

Seasonal changes in entrainment cues for the circadian rhythm of the supratidal amphipod Talorchestia longicornis

The supratidal amphipod Talorchestia longicornis Say has a circadian rhythm in activity, in which it is active on the substrate surface at night and inactive in burrows during the day. The present study determined: (1) the circadian rhythms in individual versus groups of amphipods; (2) the range of temperature cycles that entrain the circadian rhythm; (3) entrainment by high-temperature cycles versus light?:?dark cycles, and (4) seasonal substrate temperature cycles. The circadian rhythm was determined by monitoring temporal changes in surface activity using a video system. Individual and groups of amphipods have similar circadian rhythms. Entrainment occurred only to temperature cycles that included temperatures below 20°C (10–20, 15–20, 17–19, 15–25°C) but not to temperatures above 20°C (20–25, 20–30°C), and required only a 2°C temperature cycle (17–19°C). Diel substrate temperatures were above 20°C in the summer and below 20°C during the winter. Upon simultaneous exposure to a diel high-temperature cycle (20–30°C) and a light?:?dark cycle phased differently, amphipods entrained to the light?:?dark cycle. Past studies found that a temperature cycle below 20°C overrode the light?:?dark cycle for entrainment. The functional significance of this change in entrainment cues may be that while buried during the winter, the activity rhythm remains in phase with the day?:?night cycle by the substrate temperature cycles. During the summer, T. longicornis switches to the light?:?dark cycle for entrainment, perhaps as a mechanism to phase activity precisely to the short summer nights.     

The Dynamics of Adaptive Changes in the Spleen of the Hibernating Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

In hibernation season during torpor bouts, the spleen weight and the hemoglobin level, as well as the total and extracted protein contents in the spleen of the ground squirrel Spermophilus undulatus are increased when animals enter torpor and reach maximum values when the body temperature drops below 25°C. All these parameters return to the characteristic values of the euthermic animals during arousal, before the body temperature increases to 20°C. There were no significant differences in the numbers of splenocytes between ground squirrels in interbout euthermia and torpor. The minimum number of splenocytes was observed in animals that entered torpor when the core body temperature was approximately 18°C. The activity of ornithine decarboxylase, a key enzyme in polyamine synthesis, which is correlated with the functional and proliferative status of lymphoid tissue, was the same for the euthermic and summer ground squirrels and decreased monotonically during torpor. Upon arousal of the animals when body temperature was below 29°C, no resumption of the spleen ornithine decarboxylase activity was observed.     

Decapitation or starvation raise haemolymph ecdysteroid titres in the ovoviviparous female cockroach, Blaberus craniifer Burm.

1. Decapitating newly emerged Blaberus craniifer females near the prothorax severs connections between the suboesophageal and prothoracic ganglia, thus depriving them of the neuroendocrine cephalic complex (including brain and suboesophageal ganglion) and the anterior end of prothoracic glands (PGs). 2. As demonstrated by enzyme immunoassay (EIA), headless females have higher levels of ecdysteroids (ECDs) in haemolymph than starved or fed females, indicating that the neuroendocrine cephalic complex influences circulating ECD levels. 3. The time course of hormonal peaks in decapitated females resembles that in starved females during the first post-ecdysial week, suggesting that some as yet unknown regulating mechanism of ECD production lies outside the head. 4. It is suggested that: (a) The PGs are sites for ECDs production in the early post-imaginal period, (b) the prothoracic and suboesophageal ganglia (linked by nerves to PGs) regulate PGs activity, possibly via neural inputs.     

Evidence for two forms of phospholipase A2 in human semen

The molecular weight of the active unit of phospholipase A₂ (PA₂) in human seminal plasma and spermatozoa was determined using the radiation inactivation technique. Fresh spermatozoa possess more than one form of PA₂ activity as judged by the biphasic nature of the curve obtained during enzyme inactivation. However, when stored frozen for several months followed by a period of heating for 60 min at 60 °C prior to irradiation, the sperm exhibited PA₂ activity, which corresponded to a single low molecular mass form of 12,000 d when radioactive phosphatidylcholine (PC) was used as substrate and 8,000 d when radioactive phosphatidylethanolamine (PE) was used as substrate. In fresh seminal fluid, only one active form of PA₂ was detected as judged by the linear nature of the curve obtained during enzyme inactivation by irradiation. Using PC as substrate, the active unit was again estimated to be 12,000 d, whereas it corresponded to 18,000 d when PE was used. The PA₂ activity associated with normal spermatozoa exhibited a 60% decrease in activity after storage at ?20 °C for 48 hr followed by a heating period of 10 min at 60 °C. Long-term storage of spermatozoa at ?20 °C also resulted in a similar decrease in the deacylation of PC. No further loss of activity was observed during subsequent heat treatment at 60 °C. Seminal plasma, however, showed no loss of activity following short (48 hr at 4 °C or ?20 °C) or long-term storage and subsequent heat treatment. Thus, the behavior of PA₂ when the effect of temperature was studied and in radiation inactivation experiments indicates that the low molecular weight component in the seminal plasma as well as in spermatozoa is temperature resistant. However, in fresh spermatozoa, a second form of PA₂ was found and was sensitive to changes in temperature.     

Programmation de la diapause chez Gryllus campestris

Gryllus campestris has two types of development: one consists of rapid growth followed by a diapause in the penultimate larval stage, the ‘protostage’, and the other of slow growth without diapause. Experiments were designed to find the details of the programme for the two types of development.Two main temperatures, 30 and 20°C, were chosen. The sensitive period of the life cycle at 20°C was detected by subjecting insects to this temperature for a determined and increasing number of days before rearing them at 30°C during the rest of larval life. Conversely, insects were reared first at 30°C for a determined and increasing number of days and then transferred to 20°C. All these experiments led to the conclusion that the sensitive period occurs in the three last larval stages before the protostage, and that the animal is capable of following only two types of development. Experiments in which the speed of growth was slowed down by rearing insects at a cooler temperature (12°C) show that the conditions of the formation programme are not simply linked to a retardation of growth.The main effect of changing the length of days is to synchronize the arrival of all the insects at the protostage before the autumn, no matter whether the data of birth was spring or summer, or whether the number of larval stages was influenced: this explains the univoltinism of this species. Further experiments consisting of changing the length of days in the opposite direction from that of the natural seasonal evolution (increasing: 10 to 16 hr light/day instead of decreasing 16 to 10 hr) show that the programme is disturbed and subsequently the morphology of the animals is affected.To understand the genetic background of the programme a non-diapausing strain was used. It is demonstrated that selection eliminates diapause when reared at 30°C as well as normal survival when reared at 20°C. Therefore, the hypothesis of a ‘unitary system’ is here proposed for these two types of development. Hybridization of the diapausing and non-diapausing strains shows that the expression of ‘diapause’ as a response to high temperature is influenced by photoperiodism; it is dominant in short days and recessive in long days.     

Temporal occurrence of the pupal melanization reducing factor during development of the butterfly, Inachis io

Abstract. . In prepupae of Inachis io L. (Lepidoptera: Nymphalidae), a pupal melanization reducing factor (PMRF) which controls morphological colour adaptation (Bückmann & Maisch, 1987) is located in the brain, suboesophageal ganglion, thoracic ganglia, and all abdominal ganglia and their closely associated neurohaemal organs (Stamecker et al , 1994)
In animals adapted to a yellow background, PMRF content decreased in all these ganglia complexes during the prepupal stage which may be due to a release of the hormone at the critical period of the melanization reducing effect. The release of PMRF apparently occurs in a slow, but continuous, manner and may be superimposed by an incessant PMRF production at the same time recognizable by reincreasing melanization scores towards the end of prepupal and beginning of pupal stage. Therefore PMRF content in ganglia were not completely exhausted. When animals were kept on a black background, such a decline of PMRF content did not occur in both posterior ganglia complexes, whereas values from brain-suboesophageal ganglion complexes were too variable.
The target cells seem to be sensitive to PMRF treatment over a wide time range of nearly 20 h from the early stage of spinning a silk mat to 13-h-old prepupae for the melanization reducing effect.
PMRF activity was also detected in first-instar larvae and in the nervous system of third-instar larvae as well as in pupae which had completed their pigmentation. Furthermore, all three parts of the adult body still contained PMRF. Possibly PMRF may have functions in larval and adult stages in addition to its effect on morphological colour adaptation.     

No temperature acclimation of soil extracellular enzymes to experimental warming in an alpine grassland ecosystem on the Tibetan Plateau

Alpine grassland soils store large amounts of soil organic carbon (SOC) and are susceptible to rising air temperature. Soil extracellular enzymes catalyze the rate-limiting step in SOC decomposition and their catalysis, production and degradation rates are regulated by temperature. Therefore, the responses of these enzymes to warming could have a profound impact on carbon cycling in the alpine grassland ecosystems. This study was conducted to measure the responses of soil extracellular enzyme activity and temperature sensitivity (Q₁₀) to experimental warming in samples from an alpine grassland ecosystem on the Tibetan Plateau. A free air-temperature enhancement system was set up in May 2006. We measured soil microbial biomass, nutrient availability and the activity of five extracellular enzymes in 2009 and 2010. The Q₁₀ of each enzyme was calculated using a simple first-order exponential equation. We found that warming had no significant effects on soil microbial biomass C, the labile C or N content, or nutrient availability. Significant differences in the activity of most extracellular enzymes among sampling dates were found, with typically higher enzyme activity during the warm period of the year. The effects of warming on the activity of the five extracellular enzymes at 20 °C were not significant. Enzyme activity in vitro strongly increased with temperature up to 27 °C or over 30 °C (optimum temperature; T_opt). Seasonal variations in the Q₁₀ were found, but the effects of warming on Q₁₀ were not significant. We conclude that soil extracellular enzymes adapted to seasonal temperature variations, but did not acclimate to the field experimental warming.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.     

Effects of Light and Temperature on Flower-opening of Pharbitis nil

Flower buds of Pharbitis nil cut from plants growing in thefield opened rapidly when kept in darkness for 8 hr followedby continuous light at 20–25°C, but those kept indarkness for 4 hr opened promptly oniy when the temperatureduring the following light period was kept at 23°C or lower.Buds exposed to continuous light at 25°C did not open, butthose exposed to continuous light at 23°C opened slowly.At a lower temperature, the buds opened rapidly even in continuouslight. When the buds were placed in darkness at 25°C at13:30, 17:30 and 21:30 (artificial light from 17:30 to 21:30),they opened about 10 hr after the onset of darkness regardlessof the time of the onset of darkness, but when the buds werekept at 20°C in light from 13:30, 17:30 and 21:30, theyopened at 3:30–5:30 regardless of the time of transferto the lower temperature. The biological clock which controlsthe time of flower-opening is suggested to be easily reset bya light-off signal, but not by a shift from a normal to lowertemperature (20°C). At the lower temperature, the time offlower-opening probably is determined by the time of the latestpreceding light-off (or light-on) signal. ¹Dedicated to Professor Dr. Erwin Biinning on the occasion ofhis 75th birthday. (Received October 23, 1980; Accepted December 15, 1980)     

Sulfate esterifying activity of embryonic chick liver in vitro

A method has been described for the study of tissue sulfate-conjugating systems in vitro. Liver slices from embryonic chicks were maintained in vitro in a medium containing labeled inorganic sulfate and phenol. It was found that more of the sulfate was esterified at 20 °C. than at 37 °C. due to the longer continued activity at the lower temperature. All sulfate-esterifying activity was lost in liver slices maintained at 37 °C. for 30 hr. while those cultures maintained at 20 °C. continued to esterify sulfate for 70 hr.On the basis of our data there would appear to be a change in the thermal stability of the sulfate-esterifying enzyme system of the chick liver upon its transition from the embryonic stage to the stage of the fully developed chick. Data were presented for the chick 4 months ex ovo. We have been unable to detect any analogous temperature effects upon the sulfate-esterifying system in the livers of embryonic and adult rats.     

Alkaline phosphatase activity in the brain of the American cockroach Periplaneta americana L

Summary The supra- and suboesophageal ganglia of the American cockroach contain material which catalyses the alkaline hydrolysis (pH 9.5) of 5-bromo-4-chloro-3-indolyl phosphate in the presence of Nitro blue tetrazolium. Histochemical studies on unfixed cryostat sections indicate that this type of alkaline phosphatase is restricted to discrete regions in the cockroach brain. Highest enzyme activity is encountered in the mushroom bodies, central body, antennal glomeruli and specific parts of some distinct neural connections including the optic nerve, antennal nerve, circumoesophageal connectives and nerves leaving the suboesophageal ganglion. Tissue fixation by use of formaldehyde-type fixatives, as well as routine paraffin-embedding, completely destroy all histochemically detectable enzyme activity.Native polyacrylamide gradient electrophoresis suggests that the alkaline phosphatase activity is present as multiple isozymic forms, which show up in the 120–130 kD range of standard proteins. Enzyme activity becomes undetectable after fixation (trichloroacetic acid, formaldehyde containing fixatives) of electrophoretically separated native proteins, as well as after electrophoresis in denaturing conditions (SDS and -mercapto-ethanol, boiling). However, the enzyme activity remains virtually unaffected after storage of the sample for prolonged periods at –20 to –80°C.     

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, from tropical and temperate regions

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, obtained from south-central Mexico (19°N latitude) and southeast Missouri (37°N latitude) were compared. Day length and temperature were found to serve as environmental cues to programme the larval diapause of both populations, but different critical values were observed. The critical day length for diapause induction was about 13 hr light/day for Mexican larvae and about 15 hr light/day for Missouri larvae, and was relatively stable at 20 to 30°C. Mexican larvae displayed a less-intense diapause than did Missouri larvae. Some diapausing Mexican larvae maintained at 25 or 30°C pupated in about 15 days, regardless of the day length to which they were exposed. The rate of diapause development of Mexican larvae was high at day lengths between 14 hr and 16 hr, whereas that of Missouri larvae was accelerated at day lengths of 16 hr at 25 and 30°C. Diapause development of Mexican larvae was virtually unaffected by chilling at 10°C, whereas that of Missouri larvae continued at a low rate at 10°C. Selection of Mexican larvae for diapause showed that only four generations were needed to significantly increase the incidence of diapause.     

Production and partial characterization of β-1,3-glucanase obtained from Rhodotorula oryzicola

The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of β-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of β-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12?hr. During this period, 9.26 generations were obtained, with 1?hr and 29?min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr^?1). The entire microorganism growth process was monitored during β-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1?hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.     

Environmental factors controlling the time of flower-opening in Pharbitis nil

Pharbitis nil, strain Violet, subjected to various photoperiods(24-hr cycle at 24?C) bloomed about 10 hr after light-off whenthe light period was 10 hr or longer, and about 20 hr afterlight-on when the light period was shorter. The higher the temperature(20–30?C) during the dark period, the later the time offlower-opening, with the temperature during the last half ofthe dark period having a stronger effect than that during thefirst half. In continuous dark or light, flower buds of Pharbitis openedabout every 24 hr at all temperatures tested between 20 and28?C, which suggests the participation of a circadian rhythmin determining the time of flower-opening. A light pulse given6–12 or 28–36 hr after the onset of the dark periodgreatly advanced the phase of this rhythm (8–10 hr). Phasedelay of this rhythm could not be obtained by light pulses givenat any time. (Received September 29, 1979; )     

The effects of temperature and moisture on survival capacity,cuticular permeability,hemolymph osmoregulation and metabolism in the scorpion,Centruroides hentzi (banks) (scorpiones,buthidae)

• 1.1. The temperature and water relations of Centruroides hentzi females were investigated. At 12 and 72% relative humidity (RH), the lower and upper Lt₅₀ were -4.5 and 43.7°C, and -4.7 and 45.1°C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions.
• 2.2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm²/hr at 30°C to 0.211 at 41°C.
• 3.3. Exposure to dry air (0–5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period.
• 4.4. Mean oxygen consumption rates increased from 161.7 mm³/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.

     

Selection of a Photosynthetic Bacterium Suitable for Hydrogen Production in Outdoor Cultures among Strains Isolated in the Seoul,Taegu, Sendai and Bangkok Areas

Very efficient hydrogen producing photosynthetic bacteria, strains SL1, SL3, SL16 and TG28 newly isolated in Korea, and strain KM113 newly isolated in the Sendai area, were found to be Rhodopseudomonas spp. To examine the stability of cell suspensions of the cultures for hydrogen production, which is closely associated with light absorption, we conducted larger scale cultures under periodic illumination (12-hr intervals) without stirring at 30°C using strains SL1 and Rhodopseudomonas sphaeroides B5, the latter was isolated in the Bangkok area. Both strains gave homogeneous cell suspensions throughout the incubation period and larger amounts of hydrogen were produced in a shorter period of time by both cultures than obtained with Rhodopseudomonas sp. TN3, an isolate from the Sendai area which was reported previously. With the cells of the new isolates and strains TN3 and B5 grown on glutamate-malate medium at 30°C, we measured hydrogen production at 20, 30 and 40°C in the same medium. Among them, strains SL1, SL16 and KM113 showed the highest hydrogen production activity at 30°C. The maximum hydrogen production rates with these strains were over 130 µ1/hr/mg dry cells, but at 40°C, the highest activity (138 µl/hr/mg dry cells) was obtained with strain B5. Since strain B5 also showed good activities at 20 and 30°C, we suggest that this strain might be suitable for hydrogen production in outdoor cultures.     

The effect of temperature on feeding,growth, and metabolism during the last larval stadium of the female house cricket, Acheta domesticus

Eighth instar female house crickets at 35°C developed faster, gained slightly more wet weight, and consumed less food, water, and oxygen than at 25°C. The duration of the 8th stadium at 25°C was 13 days (undisturbed), but was 14 days when disturbed by daily weighing. The duration of the 8th stadium at 30°C was 8 days and at 35°C was 6 days. During the first half of the 8th stadium at 25, 30, and 35°C, there was a high rate of food and water consumption resulting in statistically equal maximum dry weight achievement (124 mg). Respiratory quotients greater than one during this time indicated the conversion of ingested carbohydrate to fat. During the latter half of the 8th stadium, food and water consumption declined and the crickets lost weight. The period of weight loss was proportionally much longer at 25°C than at 30 or 35°C. Respiratory quotients lower than 1.0 during the latter half of the 8th stadium at 30 and 35°C indicated the metabolism of stored lipids. The respiratory quotient at 25°C never fell below 1.0, possibly because some food remained in the gut. The absorption efficiency was not influenced by temperature (25–35°C). Though the caloric content of the faeces was lower at 25°C than at 30 or 35°C, which correlated to the much longer time for food passage at 25°C than at 35°C, the difference in total calories egested was insufficient to alter the absorption efficiency. A longer period of reduced feeding and greater dry weight loss during the latter half of the 8th stadium at 25°C resulted in a lower metabolic efficiency at 25°C than at 30 or 35°C. Eighth instar crickets in response to a step-function transfer from 30°C–25 or 35°C showed an immediate (<1 hr) and complete metabolic adjustment which was not affected by the temperature history during the 7th stadium. House crickets did not exhibit temperature acclimation in the range 20–40°C, the metabolic rate being determined by ambient temperature. The Q₁₀ for oxygen consumption in the range 20–40°C was about 2.     

Purification and Characterization of Thermostable Purine Nucleoside Phosphorylase of Bacillus stearothermophilus JTS 859

A thermostable purine nucleoside phosphorylase has been purified more than 800-fold from Bacillus stearothermophilus JTS 859. The enzyme had a molecular weight of 68,000 consisting of 2 identical subunits (A/_w, 34,000). The isoelectric point of the enzyme was 4.7. The enzyme did not contain cysteine. The optimal pH of the enzyme reaction was from 7.5 to 11.0. The Michaelis constants for inosine, guanosine, 2′-deoxyinosine, and 2′-deoxyguanosine were 0.22, 0.14, 0.20, and 0.10mM, respectively. The optimal temperature of the reaction was 80 C. The half-life of the enzyme was 16 hr in 20mM potassium phosphate and ImM inosine (pH 7.0) at 80°C, and no decrease of the enzyme activity was observed at least for the first 30 hr at 70°C.