Purification and Kinetic Properties of Betaine-Homocysteine Methyltransferase from Aphanothece halophytica

Betaine-homocysteine methyl transferase (BHMT) from Aphanothece halophytica was purified to homogeneity by hydroxyapatite, DEAE-Sepharose CL-6B and Sephadex G-200 column chromatography. A 24-fold purification and 11% overall yield were achieved with a specific activity of 595 nmol h⁻¹ mg⁻¹. The subunit molecular weight was determined to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the native enzyme was found to have a molecular weight of 350 kDa, suggesting an octameric structure of the enzyme. The enzyme shows optimum activity at 37°C, pH 7.5. The apparent Km values for glycinebetaine and L-homocysteine were 4.3 mM and 1.3 mM, respectively. The enzyme was 70% inactivated by 5 mM dimethylglycine whereas the same concentration of sarcosine slightly inactivated the enzyme. Two analogs of glycinebetaine were also tested for enzyme inactivation and it was found that 5 mM choline inactivated 60% of the enzyme activity and 2.5 mM betaine aldehyde completely abolished the enzyme activity. NaCl at 200 mM or higher also completely inactivated the enzyme. Received: 6 December 2000 / Accepted: 10 January 2001     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Regulation of S-adenosylhomocysteine hydrolase in the halophilic cyanobacterium Aphanothece halophytica: a possible role in glycinebetaine biosynthesis

Aphanothece halophytica, a halophilic cyanobacterium capable of growing in saturated NaCl, accumulates high intracellular concentrations of glycinebetaine in response to increasing environmental NaCl. In this organism, intracellular levels of K⁺ rise dramatically with increasing external NaCl before an increase in glycinebetaine can be detected. Glycinebetaine synthesis requires three S-adenosylmethionine (AdoMet)-mediated transmethylations; each transmethylation reaction generates one molecule of the transmethylation inhibitor S-adenosylhomocysteine (AdoHcy). Thus, glycinebetaine synthesis should require continued removal of AdoHcy. In A. halophytica, catabolism of AdoHcy was shown to occur via the reversible reaction catalyzed by AdoHcy hydrolase (EC 3.3.1.1). Activity of AdoHcy hydrolase in the direction of synthesis of AdoHcy was inhibited by 0.4 M KCl in this organism. On the other hand, activity of AdoHcy hydrolase in the direction of AdoHcy hydrolysis was unaffected by 0.4 M KCl. Glycinebetaine increased synthesis of AdoHcy in the presence of 0.4 KCl, but had no effect on AdoHcy hydrolysis. Based upon these results, a mechanism is proposed for the regulation of glycinebetaine synthesis by K⁺ and glycinebetaine in A. halophytica. According to this mechanism, the regulatory response would be initiated by a K⁺-induced shift in the AdoMet/AdoHcy ratio.Abbreviations AdoMet S-adenosylmethionine - AdoHcy S-adenosyl homocysteine     

Production of laccases in submerged process by <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis> PS-2001 in relation to carbon and organic nitrogen sources,antifoams and Tween 80

Some conditions in media composition for laccases production, such as different sources of carbon and organic nitrogen, antifoams and a surfactant, were studied in liquid cultures of Pleurotus sajor-caju strain PS-2001. Cultivation with fructose or glucose as carbon sources produced maximum enzyme activities of 37 and 36 U mL⁻¹, respectively. When sucrose was present in the medium, the best results were obtained using 5 g L⁻¹ of this carbohydrate, on the 11th day of the process, attaining laccase titres of 13 U mL⁻¹. In a medium without casein, practically no enzyme was produced during the experiments; among the sources of nitrogen studied, pure casein led to the highest titres of laccase activity. Different concentrations of pure casein and sucrose were also tested. As to the different concentrations of casein, the addition of 1.5 g L⁻¹ resulted in the highest titres of laccase activity. Negligible levels of manganese peroxidase activity were also detected in the culture medium. In low concentrations, polypropylene glycol or silicon-based antifoams and the surfactant Tween 80 have no significant influence on the formation of laccases by P. sajor-caju. However, enhanced concentration of polypropylene glycol negatively affected the production of laccases but favored the titres in total peroxidases, lignin peroxidase and veratryl alcohol oxidase.     

Production of laccase by a newly isolated deuteromycete fungus <Emphasis Type="Italic">Pestalotiopsis</Emphasis> sp. and its decolorization of azo dye

The effect of various carbon and nitrogen sources on the production of laccase by newly isolated deuteromycete Pestalotiopsis sp. was tested under liquid-state fermentation. Twenty grams per liter of glucose and 10 g l⁻¹ ammonium tartrate were found to be the optimized concentrations of carbon and nitrogen sources, respectively. The influence of different inducers and inhibitors on the laccase production was also examined. Adding the Cu up to optimum concentration of 2.0 mM in medium (include 20 g l⁻¹ glucose and 10 g l⁻¹ ammonium tartrate), the highest laccase activity of 32.7 ± 1.7 U ml^−l was achieved. Cu had to be supplemented after 2 days of growth for its maximal effect, an addition after 6 days of growth, during which laccase activity was dominantly formed, resulted in distinctly reduced laccase activity. In addition, Direct Fast Blue B2RL can be effectively decolorized by crude laccase, the decolorization percentage of which was 88.0 ± 3.2% at pH 4.0 within 12 h. The results suggest that Pestalotiopsis sp. is a high potential producer of the industrially important enzyme laccase.     

Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l⁻¹ and from 55 to 390 U l⁻¹, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.     

Kinetics of kojic acid fermentation by Aspergillus flavus using different types and concentrations of carbon and nitrogen sources

Kinetics of kojic acid fermentation by Aspergillus flavus Link 44-1 using various sources of carbon [glucose, xylose, sucrose, starch, maltose, lactose or fructose] and nitrogen [NH₄Cl, (NH₄)₂S₂O₈, (NH₄)₂NO₃, yeast extract or peptone] were analyzed using models based on logistic and Luedeking–Piret equations. The highest kojic acid production (39.90 g l⁻¹) in submerged batch fermentation was obtained when 100 g l⁻¹ glucose was used as a carbon source. Organic nitrogen sources such as peptone and yeast extract were favorable for kojic acid production as compared to inorganic nitrogen sources. Yeast extract at 5 g l⁻¹ was optimal. The optimal carbon to nitrogen (C/N) ratio for kojic acid fermentation was 93.3. In a resuspended cell system, the rate of glucose conversion to kojic acid by cell-bound enzymes increased with increasing glucose concentration up to 70 g l⁻¹, suggesting that the reaction followed the Michaelis–Menten enzyme kinetic model. The value of K _m and V _max for the reaction was 18.47 g l⁻¹ glucose and 0.154 g l⁻¹ h⁻¹, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 20–24. Received 13 October 1999/ Accepted in revised form 02 April 2000     

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l⁻¹ xylan and 10.2 g l⁻¹ yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml⁻¹ after 168-h shake culture at 4°C. In addition, very little endoglucanase, β-mannanase, β-xylosidase, β-glucosidase, α-l-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10°C in a 5-l fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0–5.5 and good stability at pH 4–9 (21 h at 4°C). Although the enzyme was maximally active at 45°–50°C, it appeared very thermolabile, showing a half-life of 78 min at 35°C. At 40°–50°C, it lost 71%–95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast. Received: December 10, 1999 / Accepted: March 23, 2000     

A process for the production of ectoine and poly(3-hydroxybutyrate) by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH₄⁺, and PO₄³⁻. In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

Carbon and Nitrogen Source Nutrition of Fumagillin Biosynthesis by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L⁻¹ xylan and 50 g L⁻¹ mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L⁻¹ was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings. Received: 8 April 2002 / Accepted: 24 June 2002     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Effect of medium osmolarity on the bioproduction of glycerol and ethanol by Hansenula anomala growing on glucose and ammonium

The osmotolerant yeast Hansenula anomala survives in media at low water activity resulting from increasing NaCl concentrations in the culture medium by producing compatible solutes. High salinity resulted in the use of a large part of the assimilated carbon substrate (glucose) for cell maintenance (28%), required for intracellular synthesis compounds and for osmotic cell regulation. The maintenance coefficient for non-growth-associated glucose consumption was found to be 0.38 mmol glucose g biomass⁻¹ h⁻¹. For decreasing water activity, there is a competition between the pathways leading to glycerol and ethanol production, until an experimental ethanol/total glycerol ratio reached a value 3.4 for 2 mol l⁻¹ NaCl (close to the theoretical value of 4)—illustrating the osmodependent channelling of carbon towards polyols production. This competition leads to a cessation of ethanol production during the stationary state before that of glycerol. Since osmotic adjustment occurred mainly during growth, glycerol production during stationary state can be clearly related to another mechanism other than osmotic: it was excreted by a fermentative mechanism to ensure energy for cell maintenance.     

Production and characterization of esterase and lipase from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis>

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h⁻¹ and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l⁻¹ using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h⁻¹ (at 42.5°C, pH 7.4, and 3.6 mol l⁻¹ NaCl), 2.3 U l⁻¹ (at 50°C, pH 7.5, and 4.3 mol l⁻¹ NaCl), and 0.58 U l⁻¹ (at 50°C, pH 7.6, and 4.5 mol l⁻¹ NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l⁻¹ NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.     

Effects of salinity on growth and compatible solutes of callus induced from <Emphasis Type="Italic">Populus euphratica</Emphasis>

Summary The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.     

Sucrose enhances phosphoenolpyruvate carboxylase activity of in vitro solanum tuberosum L. under non-limiting nitrogen conditions

Summary The effect of sucrose on in vitro potato (ev. Kennebec) metabolism was evaluated. Plants were grown in three different media: Murashige and Skoog basal medium containing high nitrogen concentration with 0 or 20 g l⁻¹ sucrose; or modified medium containing reduced nitrogen amount and 20 g l⁻¹ sucrose. Plants fed with 20 g l⁻¹ sucrose and high N exhibited higher phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase activities and high PEPC protein concentration at 7, 20 and 33 d of culture compared to those grown with 20 g l⁻¹ sucrose and low N, or with 0 g l⁻¹ sucrose and high nitrogen (control). The highest accumulation of starch and sucrose was found in plants grown with sucrose and low nitrogen. This accumulation occurred concomitantly with a reduced enzyme activity resulting from a low utilization of α-ketoglutarate by nitrogen assimilation, when plants were grown with reduced nitrogen. Our investigations on tricarboxylic acid cycle activity showed that sucrose led to the reduction of organic acid amounts in both leaves and roots when high nitrogen was supplied to plants. This was probably due to the intense exit of α-ketoglutarate, which was confirmed by measurements of cytosolic isocitrate dehydrogenase activity. The low leaf glutamine/glutamate ratio observed in plants grown with 20 g l⁻¹ sucrose and high nitrogen compared to their counterparts cultivated with low nitrogen might be due to glutamine conversion into proteins when nitrogen assimilation was intense. These results demonstrate that sucrose enhanced PEPC activity by increasing protein synthesis. They also suggest that sucrose metabolism is involved in the replenishment of the tricarboxylic acid cycle by providing carbon skeletons required to sustain phosphoenolpyruvate utilization during high nitrate assimilation.     

Effects of salts on activity of halophilic cellulase with glucomannanase activity isolated from alkaliphilic and halophilic Bacillus sp. BG-CS10

Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The K _m and k _cat values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL⁻¹ and 26 s⁻¹, while the K _m and k _cat values of CelB without NaCl were 6.6 mg mL⁻¹ and 2.1 s⁻¹. Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant <Emphasis Type="Italic">Thermus thermophilus</Emphasis> RQ-1

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70°C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77°C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 μmol/mg protein compared to batch fermentations (0.31 μmol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l⁻¹ h⁻¹. The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70°C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.     

Responses of antioxidant defense system of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don. to paclobutrazol treatment under salinity

The effect of paclobutrazol, a plant growth regulator, on antioxidant defense system was investigated in Catharanthus roseus (L.) G. Don. plants subjected to NaCl stress. The growth parameters were significantly reduced under 80 mM NaCl treatment; however, this growth inhibition was less in paclobutrazol-treated (15 mg l⁻¹ plant⁻¹) plants. The non-enzymatic antioxidants ascorbic acid and reduced glutathione were affected under NaCl stress and they increased significantly under paclobutrazol treatment when compared to NaCl treated as well as control plants (P ≤ 0.05). The activity of antioxidant enzyme ascorbate peroxidase showed a significant enhancement under salinity stress. The catalase activity decreased in roots of NaCl-treated plants, but recovered with paclobutrazol treatment. The results suggested that paclobutrazol have significant role in contributing salt stress tolerance of C. roseus by improving the components of antioxidant defense system.