Biotransformation of Isoeugenol to Vanillin by a Novel Strain of <Emphasis Type="Italic">Bacillus fusiformis</Emphasis>

A novel strain of Bacillus fusiformis, producing high amounts of vanillin from isoeugenol, was isolated from soil. Using 60% (v/v) isoeugenol as substrate and solvent and at pH 4.0, 37 °C and 180 rpm, vanillin was produced at 32.5 g l⁻¹ over 72 h. The unused isoeugenol was reusable.     

Physiological differences in the growth of <Emphasis Type="Italic">Sargassum horneri</Emphasis> between the germling and adult stages

The effects of temperature, irradiance, and daylength on Sargassum horneri growth were examined at the germling and adult stages to discern their physiological differences. Temperature–irradiance (10, 15, 20, 25, 30°C × 20, 40, 80 μmol photons m⁻²s⁻¹) and daylength (8, 12, 16, 24 h) experiments were carried out. The germlings and blades of S. horneri grew over a wide range of temperatures (10–25°C), irradiances (20–80 μmol photons m⁻²s⁻¹), and daylengths (8–24 h). At the optimal growth conditions, the relative growth rates (RGR) of the germlings were 21% day⁻¹ (25°C, 20 μmol photons m⁻²s⁻¹) and 13% day⁻¹ (8 h daylength). In contrast, the RGRs of the blade weights were 4% day⁻¹ (15°C, 20 μmol photons m⁻²s⁻¹) and 5% day⁻¹ (12 h daylength). Negative growth rates were found at 20 μmol photons m⁻²s⁻¹ of 20°C and 25°C treatments after 12 days. This phenomenon coincides with the necrosis of S. horneri blades in field populations. In conclusion, we found physiological differences between S. horneri germlings and adults with respect to daylength and temperature optima. The growth of S. horneri germlings could be enhanced at 25°C, 20 μmol photons m⁻²s⁻¹, and 8 h daylength for construction of Sargassum beds and restoration of barren areas.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Degradation of diphenyl phthalate by <Emphasis Type="Italic">Sphingomonas chungbukensis</Emphasis>

More than 80% of diphenyl phthalate (DPP) at 100 mg l⁻¹ was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30°C within 48 h. The maximum specific degradation rate was 5 mg DPP l⁻¹ h⁻¹. It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.     

Galactooligosaccharide production by a thermostable β-galactosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal at pH 6.0 and 85°C. The half-lives of the recombinant β-galactosidase at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and 2.4 h, respectively, and its deactivation energy was 213 kJ mol⁻¹. The optimal amount of enzyme for effective GOS production was 3.6 U of enzyme ml⁻¹. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant. The rates of hydrolysis and transgalactosylation reactions increased with increasing temperature but the final concentration of GOS was maximal at 80°C. Under the conditions of pH 6.0, 80°C, 600 g lactose l⁻¹, and 3.6 U enzyme ml⁻¹, 315 g GOS l⁻¹ were obtained for 56 h with a yield of 52.5% (w/w). The β-galactosidase from S. solfataricus produced GOS with the highest concentration and yield among thermostable β-galactosidases reported to date.     

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.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp

A new basidiomycete, Trametes sp. 420, produced laccase at 6,810 U l⁻¹ (268 mg, 25.4 U mg⁻¹ protein for guaiacol) in glucose medium and 7,870 U l⁻¹ (310 mg) in cellobiose medium with induction by 0.5 mM Cu²⁺ and 6 mM o-toluidine. Laccase isozyme E (LacE) was the sole laccase in the fermentation products. It was stable at pH 5–9 and below 70°C over 30 min. The K _m values of LacE for four substrates (guaiacol ABTS, 2,6-dimethoxyphenol and syringaldazine) varied from 5 to 245 μM. The activity of LacE was strongly inhibited by NaN₃ but not by EDTA or dimethylsulfoxide. LacE at 0.5 U l⁻¹ could decolorize industrial dyes. The open reading frame of the lacE gene was 2,130 bp and was interrupted by 10 introns. It displayed a high homology to laccases from other fungi. Pingui Tong and Yuzhi Hong contributed equally to the study     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Biosynthesis and properties of an extracellular thermostable serine alkaline protease from <Emphasis Type="Italic">Virgibacillus pantothenticus</Emphasis>

In this communication, we report the presence of a newly identified serine alkaline protease producing bacteria, Virgibacillus pantothenticus (MTCC 6729) in the fresh chicken meat samples and the factors affecting biosynthesis as well as characterization of protease. The strain produced only 14.3 U ml⁻¹ protease in the standard medium after 72 h of incubation, while in optimized culture conditions the production of protease was increased up to 18.2 U ml⁻¹. The strain was able to produce protease at 40°C at pH 9.0. The addition of dextrose and casein improved protease production. The protease was partially purified and characterized in terms of pH and temperature stability, effect of metal ions and inhibitors. The protease was found to be thermostable alkaline by retaining its 100% and 85% stability at pH 10.0 and at 50°C respectively. The protease was compatible with some of the commercial detergents tested, and was effective in removing protein stains from cotton fabrics. The V. pantothenticus, MTCC 6729 protease appears to be potentially useful as an additive in detergents as a stain remover and other bio-formulations.     

Optimal activity and thermostability of xylose reductase from <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170

Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55°C, respectively, was evaluated by a 2² central composite design face-centered. The F-test (ANOVA) and the Student’s t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively. The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL⁻¹, corresponding to 0.07–0.352 U mg⁻¹, whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R ² = 0.940) maximum volumetric activity of 2.27 U mL⁻¹ and specific activity of 0.300 U mg⁻¹ at pH 5.3 and 39°C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very stable at low temperature (4°C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at 39°C. On the other hand, at temperatures ≥50°C it was lost almost completely after only 20 min.     

A novel alkalo- and thermostable phospholipase D from <Emphasis Type="Italic">Streptomyces olivochromogenes</Emphasis>

A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75°C and the enzyme was stable from pH 7 to 13 and from 55 to 75°C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca²⁺-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 μmol min⁻¹ mg⁻¹, respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50°C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Purification and characterization of a novel endo-β-1,4-glucanase from the thermoacidophilic <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

An endo-β-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11, was purified 18-fold with 14% yield and a specific activity of 67 U mg⁻¹ protein. The optimal pH was 2 and the cellulase was stable from pH 2 to 5. The cellulase had a temperature optimum of 60°C measured over 30 min and retained more than 60% of its activity after heating at 70°C for 1 h. The molecular mass of the cellulase was about 25 kDa. Its activity was inhibited by 77% by Hg²⁺ (2 mM) and by 59% by Cu²⁺ (2 mM).     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

Phenology of Chondrus ocellatus in Cheongsapo Near Busan, Korea

The reproductive phenology of Chondrus ocellatus and the effects of temperature and light on its growth were examined in Cheongsapo near Busan, Korea, from September 1994 to August 1995. The vegetative plants dominated over the year, with a peak occurrence in January. Gameto- and tetrasporophytes were most abundant in November and August. All vegetative and reproductive plants had a peak both in length and weight in October, when seawater temperature was highest (24°C). In laboratory culture, the maximum relative growth rate (RGR) of 2.94% day⁻¹ was obtained at 20°C and 100 μmol photons m⁻² s⁻¹, whereas the lowest value was recorded at 25°C and 100 μmol photons m⁻² s⁻¹ in a 12: 12 h LD photoperiod regime. Among the three photoperiod regimes (8:16 h, 12:12 h, 16:8 h LD) tested, there was evidence of a higher RGR in the 12:12 h LD cycle. This result suggests that the growth and reproduction of C. ocellatus are correlated with the seawater temperature based on laboratory culture and field observations.