Immobilization of Thermomyces lanuginosus Lipase by Different Techniques on Immobead 150 Support: Characterization and Applications

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40 % when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60 % in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

Amylase Production by Saccharomycopsis fibuligera A11 in Solid-State Fermentation for Hydrolysis of Cassava Starch

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD_600 nm = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 °C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.     

Microwave Pretreatment of Substrates for Cellulase Production by Solid-State Fermentation

The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.     

Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D0, was higher for the active TLL compared to the inactive (D0 = 17.9 x 10(-11) cm2/s vs D0 = 4.1 x 10(-11) cm2/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.     

Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145

The production of cellulase-free end oxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated insemisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncobas the carbon source. The best results were with the following conditions: grain size=4.5 mm, solid:liquid ratio=1:2, and inoculum size=20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75°C, displaying a high thermostability. The apparent K ₂₅ and V _max were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.     

Characterization of Biocatalysts Prepared with Thermomyces lanuginosus Lipase and Different Silica Precursors,Dried using Aerogel and Xerogel Techniques

The use of lipases in industrial processes can result in products with high levels of purity and at the same time reduce pollutant generation and improve both selectivity and yields. In this work, lipase from Thermomyces lanuginosus was immobilized using two different techniques. The first involves the hydrolysis/polycondensation of a silica precursor (tetramethoxysilane (TMOS)) at neutral pH and ambient temperature, and the second one uses tetraethoxysilane (TEOS) as the silica precursor, involving the hydrolysis and polycondensation of the alkoxide in appropriate solvents. After immobilization, the enzymatic preparations were dried using the aerogel and xerogel techniques and then characterized in terms of their hydrolytic activities using a titrimetric method with olive oil and by the formation of 2-phenylethyl acetate in a transesterification reaction. The morphological properties of the materials were characterized using scanning electron microscopy, measurements of the surface area and pore size and volume, thermogravimetric analysis, and exploratory differential calorimetry. The results of the work indicate that the use of different silica precursors (TEOS or TMOS) and different drying techniques (aerogel or xerogel) can significantly affect the properties of the resulting biocatalyst. Drying with supercritical CO₂ provided higher enzymatic activities and pore sizes and was therefore preferable to drying, using the xerogel technique. Thermogravimetric analysis and differential scanning calorimetry analyses revealed differences in behavior between the two biocatalyst preparations due to the compounds present.     

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.     

Integrated Functional-Omics Analysis of Thermomyces lanuginosus Reveals its Potential for Simultaneous Production of Xylanase and Substituted Xylooligosaccharides

Applied Biochemistry and Biotechnology - Thermophiles have several beneficial properties for the conversion of biomass at high temperatures. Thermomyces lanuginosus is a thermophilic filamentous...     

无溶剂体系中脂肪酶催化红花油水解反应

无溶剂体系中脂肪酶催化红花油水解反应;水解;红花油;无溶剂体系;脂肪酶     

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00?±?0.15 and 0.21?±?0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH?7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.     

Precursor Supply Strategy for Tetramethylpyrazine Production by Bacillus Subtilis on Solid-State Fermentation of Wheat Bran

Tetramethylpyrazine (TTMP) is a widely used flavoring additive with a nutty and roasted taste. Solid-state fermentation (SSF) of wheat bran for producing TTMP was studied with Bacillus subtilis CCTCC M208157, which was an exogenous precursor-independent TTMP-producing strain. Factors influencing endogenous precursor supply and TTMP formation in this strain were investigated. According to the findings, glucose and diammonium phosphate contributed to TTMP production but excess salts caused an inhibition on cell growth and TTMP formation. Then a two-step supply strategy was applied: 10 % glucose was added at the beginning of the process to allow acetoin formation, which was the precursor of TTMP, while 3 % diammonium phosphate was added only after acetoin accumulation reached its maximum. By applying this strategy, acetoin increased from 5.44 to 13.2 g/kg dry substrate (kgds), and consequently the yield of TTMP increased by 6.8 folds from 0.44 to 3.01 g/kgds. This was the first report of using a two-step supply strategy for TTMP production by SSF, which proved to be conducive to TTMP production in this strain.     

Effect of Biosurfactants on Laccase Production and Phenol Biodegradation in Solid-State Fermentation

The effects of two biosurfactants, tea saponin (TS) and rhamnolipid (RL), on the production of laccase and the degradation of phenol by P. simplicissimum were investigated in solid-state fermentation consisting of rice straw, rice bran, and sawdust. Firstly, the effects of phenol on the fermentation process were studied in the absence of surfactants. Then, a phenol concentration of 3 mg/g in the fermentation was selected for detailed research with the addition of biosurfactants. The results showed that TS and RL at different concentrations had stimulative effects on the enzyme activity of laccase. The highest laccase activities during the fermentation were enhanced by 163.7%, 68.2%, and 23.3% by TS at concentrations of 0.02%, 0.06%, and 0.10%, respectively. As a result of the enhanced laccase activity, the efficiency of phenol degradation was also improved by both biosurfactants. RL caused a significant increase of fungal biomass in the early stage of the fermentation, while TS had an inhibitory effect in the whole process. These results indicated that RL could mitigate the negative effects of phenol on fungal growth and consequently improve laccase production and phenol degradation. TS was potentially applicable to phenol-polluted solid-state fermentation.     

Monitoring the Hydrolysis of Olive Oil Catalyzed by Lipase via Acid Value Detection

IntroductionThe lipase-catalyzed hydrolysis of oils is widelyused for the preparation of glycerides and fatty acidsbecause of the substrate specificity of lipase[1—3]. Thereaction is carried out under mild conditions.Candidalipolyticalipase is a nonspeci…     

Solid-State Fermentation for Humic Acids Production by a Trichoderma reesei Strain Using an Oil Palm Empty Fruit Bunch as the Substrate

Empty fruit bunch (EFB), an underutilized waste product of oil palm processing, was studied as a substrate for the production of humic acids (HA) by a Trichoderma reesei strain by solid-state fermentation (SSF) in Raimbault columns. HA have attracted the attention of many investigators due to their applications in agriculture, industry, the environment, and biomedicine. Commercial HA are currently chemically extracted from peat and coal, which are nonrenewable carbon sources. Biotechnological processes are important for their sustainable and controlled production, with SSF being especially promising for mimicking the natural habitat of fungi. Trichoderma sporulation and HA production are related, and the results of this study showed that SSF stimulated fast sporulation. The productivity related to HA was much higher than that of the biomass, indicating an efficient utilization of EFB. These findings, added to the low cost of EFB, make SSF an attractive process for HA production.     

Cellulase Production Under Solid-State Fermentation by Trichoderma reesei RUT C30: Statistical Optimization of Process Parameters

Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box-Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 degrees C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Production of Alkaline Protease by Bacillus altitudinis GVC11 using Castor Husk in Solid-State Fermentation

Castor (Ricinus communis L.) is an important oil seed crop having its main cultivated area in India, China, and Brazil in dry land farming. Castor husk is generated as waste in castor oil production. Use of castor husk waste as substrate is studied for alkaline protease production by Bacillus altitudinis GVC11 in solid-state fermentation. Various parameters like moisture content, incubation period, particle size, effect of carbon and nitrogen sources are studied and optimized for enzyme production. Highest enzyme production of 419,293 units per gram husk is obtained. Cost of enzyme production can be reduced by using castor husk as substrate.     

Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70 °C, with a 3.0 KJ mol⁻¹ increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6 U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3 U/mL, followed by cassava starch and corn starch with 5.9 and 3.0 U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose–Sepharose affinity chromatography; the estimated molecular mass was 100 kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65 °C.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.