Application of xylanase from thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse

Xylanases have significant current and potential uses for several industries including paper and pulp, food, and biofuel. For the biofuel industry, xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). We investigated the thermophilic fungus Thermomyces lanuginosus was yielded for xylanase production and found that the highest activity (850 U/mL) was yielded after 96 h of semisolid fermentation. The enzyme was used for hydrolyzing agricultural residues with and without pretreatment. Such residues were characterized in relation to the maximum xylose content by total acid hydrolysis. The highest xylose yields realized by enzymatic hydrolysis were 24 and 52%, achieved by using 3000 U/g (dried material) of sugarcane bagasse and corncob, respectively, which received both alkali and thermal pretreatment.     

Potential Application of Alkaline Pectinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SS in Pulp and Paper Industry

Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 degrees C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 degrees C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.     

Pulp bleaching using laccase from trametes versicolor under high temperature and alkaline conditions

Kraft pulp was delignified using laccase produced by the white rot fungusTrametes versicolor immobilized in solid support under specific conditions. The stability tests showed that this enzyme was stable for 6 h at 55°C and pH 8.0, allowing its use under pH and temperature conditions very close to those used in industrial bleaching. In this work, unbleached hardwood Kraft pulp was submitted to prebleaching using 2 U laccase/g pulp basis. Reaction time, temperature, and pH of the enzymatic treatment were investigated. Good results regarding Kappa number reduction, selectivities, and high viscosities were obtained when prebleaching was performed for 1 h at temperature of 55©C and pH 8.0 followed by alkaline extraction and ECF bleaching sequences.

     

Laccase from Trametes versicolor

The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp. Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used. The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C.     

Lignin peroxidase production by Streptomyces viridosporus T7A

Lignin peroxidase (LiP) production cost should be reduced to justify its use in the control of environmental pollution. In this work, we studied the enzyme production by Streptomyces viridosporus T7A using glucose or corn oil as a carbon source having 0.65% yeast extract as a nitrogen source. Enzyme activity, observed using either 0.65% glucose or corn oil at 0.1, 0.5, and 1.0% concentration, was 300, 150, 300, and 200 U/L, respectively. Although higher enzyme activity was obtained in both media containing 0.65% glucose and 0.5% corn oil, the use of corn oil resulted in a better LiP stability. When combined carbon sources were used, higher values of enzyme activity (360, 350, and 225 U/L) were observed in media with 0.65% glucose and supplemented with 0.1, 0.5, and 1.0% corn oil, respectively. Although the presence of both glucose and 0.5% corn oil is favorable for LiP production, satisfactory results in terms of enzyme production and stability could be also observed using 0.5% corn oil as a sole carbon source, which may lead to reduced production costs of the LiP enzyme.     

Production of extracellular xylanases byPenicillium janthinellum

Xylanase production byPenicillium janthinellum using 10–100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 Μmol/ min (11.4 U/mL or 84.4 U/mg) of Β-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). Β-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 Β- xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No Β-xylosidase or Β- glucosidase was induced until d 5. The Β-xylanases were rapidly inactivated at 50‡C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, theΒ-xylosidase andΒ-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.     

l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

Lipase production byPenicillium restrictum in a bench-scale fermenter

A preliminary screening work selectedPenicillium restrictum as a promising micro-organism for lipase production. The physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate (Qa) and agitation (N) was evaluated in a 5-L bench-scale fermenter. In optimized conditions for lipase production meat peptone at 2% (w/v) and olive oil at 1% (w/v) were used in a growth medium with a C/N ratio of 9.9. Higher C/N ratios favored cell growth in detriment of enzyme production. Low extracellular lipase activities were observed using glucose as carbon source suggesting glucose regulation. Final lipase accumulation of 13,000 U/L was obtained, using optimized specific air flow rate (Qa) of 0.5 wm and an impeller speed (N) of 200 rpm. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil as carbon source.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation

Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity of 5791 U/h.     

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.     

Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation

Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

Purification and Application of a Lipase from <Emphasis Type="Italic">Penicillium expansum</Emphasis> PED-03

An extracellular lipase was purified from the fermentation broth of Penicillium expansum PED-03 by DEAE-Sepharose chromatography, followed by sephacryl S-200 chromatography. The enzyme was purified 81.8-fold with 19.8% recovery and a specific activity of 85.94 U/mg. The molecular weight of the homogeneous enzyme was about 28 kDa, determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzymatic resolution of racemic ibuprofen was carried out by the lipase from P. expansum PED-03, and the conversion reached 46% with excellent enantioselectivity(E > 200 ), which showed a good application potential in the production of optically pure ibuprofen.     

Evaluation of cell recycle on Thermomyces lanuginosus xylanase a production by Pichia pastoris GS 115

This work aims to evaluate cell recycle of a recombinant strain of Pichia pastoris GS115 on the Xylanase A (XynA) production of Thermomyces lanuginosus IOC-4145 in submerged fermentation. Fed-batch processes were carried out with methanol feeding at each 12h and recycling cell at 24, 48, and 72 h. Additionally, the influence of the initial cell concentration was investigated. XynA production was not decreased with the recycling time, during four cell recycles, using an initial cell concentration of 2.5 g/L. The maximum activity was 14,050 U/L obtained in 24h of expression. However, when the initial cell concentration of 0.25 g/L was investigated, the enzymatic activity was reduced by 30 and 75% after the third and fourth cycles, respectively. Finally, it could be concluded that the initial cell concentration influenced the process performance and the interval of cell recycle affected enzymatic production.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.