Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Oxygen limitation improves glycerol production by Candida krusei in a bioreactor

An oxygen limitation strategy based on dynamic enzyme activity was applied to improve glycerol accumulation and decrease the residual sugar level in a fermentation of Candida krusei in a bioreactor. By applying oxygen limitation at 88 h when the activities of two glycerol synthetic enzymes cytosolic glycerol-3-phosphate dehydrogenase (ctGPD) and glycerol-3-phosphatase (GPP) were low and the activity of mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD) which catalyzes the glycerol dissimilation was high, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 51.8 g l⁻¹ at 96 h and 54.9 g l⁻¹ at 116 h, which was 18 and 60% higher than the control (without oxygen limitation), respectively. The residual sugar was consumed completely while it was 11.2 g l⁻¹ at the end of fermentation in the control. Under oxygen limitation, ethanol production was detected at a final concentration of 3.6 g l⁻¹. This work suggests a metabolic flux shift by oxygen limitation in the bioreactor.     

Cadmium biosorption on Spirulina platensis biomass

Dry biomass of Spirulina platensis re-hydrated for 48 h was employed as a biosorbent in tests of cadmium(II) removal from water. Various concentrations of biomass (from 1 to 4 g l⁻¹) and metal (from 100 to 800 mg l⁻¹) were tested. Low biomass levels (X_o  2 g l⁻¹) ensured metal removal up to 98% only at Cd₀= 100 and 200 mg l⁻¹, while X_o  2.0 g l⁻¹ were needed at Cd₀ = 400 mg l⁻¹ to achieve satisfactory results. Whereas X_o = 4.0 g l⁻¹ was effective to remove up to Cd₀ = 500 mg l⁻¹, a further increase in metal concentration (Cd₀ = 600 and 800 mg l⁻¹) led to progressive worsening of the system performance. At a given biomass levels, the kinetics of the process was better at low Cd²⁺ concentrations, while, raising the adsorbent level from 1.0 to 2.0 g l⁻¹ and then to 4.0 g l⁻¹, the rate constant of biosorption increased by about one order of magnitude in both cases and the adsorption capacity of the system progressively decreased from 357 to 149 mg g⁻¹.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Bioreactor operation for transgenic Nicotiana tabacum cell cultures and continuous production of recombinant human granulocyte-macrophage colony-stimulating factor by perfusion culture

The production of hGM-CSF was investigated in both a flask and a 5-l bioreactor, using transgenic Nicotiana tabacum suspension cells. While the maximum cell density and secreted hGM-CSF in the flask were 15.4 g l⁻¹ and 6.5 μg l⁻¹, respectively, those in the bioreactor were 15.6 g l⁻¹ and 7.6 μg l⁻¹. No detectable growth inhibition, shorter production of hGM-CSF and reduced cell viability in the batch bioreactor were observed under the specific conditions used compared with the flask culture. To improve the productivity, a perfusion culture was carried out in the bioreactor, with three different perfusion rates (0.5, 1.0 and 2.0 day⁻¹). In all cases, the hGM-CSF in the medium was significantly increased during the overall culture period (16 days), with maximum values 3.0-, 9.4- and 6.0-fold higher than those obtained in the batch cultures, respectively, even though the intracellular hGM-CSF content was not significantly varied by the perfusion rate. In terms of the total amount of hGM-CSF secreted, 205.5, 1073.2 and 1246.3 μg accumulated in the perfusate within 16 days at the perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. It was concluded that the beneficial effect of perfusion on the production of hGM-CSF originated from the reduced proteolytic degradation due to the lower protease activity caused by the perfusion. Additionally, the cell growth and physiology in the perfusion culture were somewhat negatively affected by the increased perfusion rate, although the dry cell density steadily increased, and as a result, 19.4, 22.4 and 22.9 g l⁻¹ of maximum cells were obtained with perfusion rates of 0.5, 1.0 and 2.0 day⁻¹, respectively. This work highlighted the importance of proteolytic degradation in plant cell cultures for the production of secretory proteins and the feasibility of perfusion strategies for the continuous production of foreign proteins by the prevention of protein loss due to proteolytic enzymes.     

Determination of kinetic constants of hybrid textile wastewater treatment system

The present study is related to treatment of textile wastewater in microaerophilic–aerobic hybrid reactor. The study showed the effectiveness of biological treatment of wastewater involving appropriate microorganism and suitable reactors. COD and color were reduced to 82–94%, and 99% respectively for textile wastewater. The reactor was operated at highest loading of 16.4 g COD g l⁻¹ d⁻¹ and obtained 80% COD and 72% color removal. Biokinetic models were applied to data obtained from experimental studies in continuously operated hybrid reactor. Treatment efficiencies of the reactor were investigated at different hydraulic retention times (2.3–9.1 d) and organic loading rates (2.6–16.4 g COD l⁻¹ d⁻¹). Second-order and a Stover–Kincannon models were best fitted to the hybrid column reactor. The second-order substrate removal rate constant (k_2(S)) was found as 41.44 d⁻¹ for hybrid reactor. Applying the modified Stover–Kincannon model to the hybrid reactor, the maximum removal rate constant (U_max) and saturation value constant (K_B) were found to be 212 g l⁻¹ d⁻¹ and 22.89 g l⁻¹ d⁻¹, respectively.     

Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens

When cultivated in Murashige & Skoog medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid and 0.5 mg l⁻¹ 6-benzyladenine, Perilla frutescens cells in suspension culture grew rapidly reaching about 13.6 g dry wt l⁻¹ after 12 days. The cell line produced both anthocyanin 0.9 g l⁻¹ and triterpenoids: 16 mg l⁻¹ oleanolic acid (OA), 25 mg l⁻¹ ursolic acid (UA) and 14 mg l⁻¹ tormentic acid (TA). When P. frutescens cells of 7-day-old cultures were exposed to a yeast elicitor at 0.5–5% (v/v) for 7 days, it was found that anthocyanin content peaked at 10.2% of dry weight with yeast elicitor at 1% (v/v) whereas the maximum production of oleanolic acid and ursolic acid in cultures treated with 2% (v/v) yeast elicitor was 19 and 27 mg l⁻¹, a 46 and 24% increase over the control, respectively. This is the first report of simultaneous production of both anthocyanin and triterpenoids in a single culture system.     

Electrophoretic and enzymatic studies on the crude extracellular enzyme system of the cellulolytic bacterium Cellulomonas sp. ATCC 21399

Cellulomonas sp. ATCC 21399 produced extracellular enzyme activities against Avicel, H(3)PO(4)-swollen Avicel, carboxymethylcellulose, (1-3, 1-4)-beta-D-heteroglucan, xylan, galactomannan, and amylose drying growth on microcrystalline cellulose. No extracellular cellobiase activity was produced. Crossed immunoelectrophoresis of the crude extracellular enzyme system revealed 15 immunologically distinct immunoprecipitates. The immunoprecipitates of endoglucanase A, endoglucanase B and the xylanase appeared heterogeneous with several optima, whereas the immunoprecipitates of endoglucanase C and the amylase appeared homogeneous. The heterogeneity of endoglucanase A, endoglucanase B and xylanase was also visualized using electrofocusing-immunoelectrophoresis. Electro-focusing could resolve the activity against carboxymethylcellulose into six peaks, whereas only one peak of activity against Avicel was observed. The later peak coincided with the major peak of activity against carboxymethylcellulose with isoelectric point between pH 4.0-5.0.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

Hydrolysis and fermentation of brewer's spent grain by Neurospora crassa

In this study, the ethanol production by the mesophilic fungus Neurospora crassa from BG was studied and optimized concerning the induction of lignocellulose degrading enzymes and the production phase as well. The production of cellulolytic and hemicellulolytic enzymes was studied under solid-state cultivation (SSC). SSC in a laboratory horizontal bioreactor using the optimized medium, WS and BG in the ratio 1:1 and initial moisture level 61.5%, allowed the large scale production of the multienzymatic system. Similar yields with those from flasks experiments, as high as 1073, 56, 4.2, 1.6, 3.1, 5.7 and 0.52 U g⁻¹ carbon source of xylanase, endoglucanase, cellobiohydrolase, β-glucosidase, -l-arabinofuranosidase, acetyl esterase and feruloyl esterase, respectively, were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl substrates were used to characterize the major activities of the multienzyme component, after the separation by isoelectric focusing (IEF) electrophoresis. Alkali pre-treated BG was used for ethanol production. A yield of about 74 g of ethanol kg⁻¹ dry BG (5, 6 g L⁻¹) was obtained under optimum conditions (aeration 0.1 vvm, pre-treatment with 1 g NaOH 10 g⁻¹ dry BG).     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Batch and continuous fermentation of succinic acid from wood hydrolysate by Mannheimia succiniciproducens MBEL55E

Batch and continuous cultures of Mannheimia succiniciproducens MBEL55E were carried out in a complex medium containing a NaOH-treated wood hydrolysate for the production of succinic acid. The wood hydrolysate based medium was treated with NaOH before sterilization to reduce the formation of inhibitory compounds. M. succiniciproducens MBEL55E utilized xylose as well as glucose in the wood hydrolysate based medium as a carbon source for the succinic acid production. In batch cultures, the final succinic acid concentration of 11.73 g l⁻¹ was obtained from the pre-treated wood hydrolysate based medium, resulting in a succinic acid yield of 56% and a succinic acid productivity of 1.17 g l⁻¹ h⁻¹, while the corresponding continuous cultures gave the succinic acid yield and productivity of 55% and 3.19 g l⁻¹ h⁻¹, respectively. These results suggest that succinic acid can be produced economically and efficiently by the fermentation of M. succiniciproducens MBEL55E from an inexpensive biomass-based wood hydrolysate.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

Xylanase production by Trichoderma harzianum E58

Summary Growth of Trichoderma harzianum E58 on hemicellulose-rich media, both in batch and fermentor cultures, resulted in independent profiles for the production of xylanase and endoglucanase enzymes. Dramatic differences in the ratio of xylanase to endoglucanase activities were observed among cultures grown on cellulose-rich Solka Floc and xylan. These results indicated that the induction of xylanases and cellulases was likely to be under separate regulatory control. The specific activity and amount of xylanases produced were found to be dependent on the concentration of xylan in the growth media. Growth on oat spelts xylan or the hemicellulose-rich, water-soluble fraction from steam-treated aspenwood (SEA-WS) greatly enhanced the production of xylanases and xylosidase in the culture filtrates. Constitutive levels of xylanase and endoglucanase enzymes were detected during growth of the fungus on glucose.Offprint requests to: D. J. Senior     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399

Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.