Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Optimization,Purification, and Characterization of Extracellular Mesophilic Alkaline Cellulase from Sponge-Associated <Emphasis Type="Italic">Marinobacter</Emphasis> sp. MSI032

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 °C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn²⁺ ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27–35 °C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.     

Aspergillus fumigatus Thermophilic and Acidophilic Endoglucanases

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer’s spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 °C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L^-1 and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 °C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g^-1 of dry substrate (wheat bran and sugarcane bagasse) within 3 days.     

Bioconversion of cellulose and simultaneous production of thermoactive exo- and endoglucanases by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Saccharification of cellulose is a promising method for production of biofuels. However, low bioconversion efficiency of cellulose to soluble sugars is a major challenge. In this study, a cellulolytic strain of Fusarium oxysporum was cultivated on pure cellulosic substrates (avicel, α-cellulose, carboxymethylcellulose and methylcellulose) and conversion efficiency into glucose was investigated. Production of exo- and endoglucanases during the bioconversion process was evaluated. Influence of pH on saccharification of cellulose and enzyme production by F. oxysporum were determined. Highest yield of glucose (1.76 μmol/ml) was obtained from F. oxysporum on methyl cellulose at 192 h under basal conditions. Liberated glucose under optimized condition of pH 6.0 at 96 h of fermentation was 2.12 μmol/ml with maximum production of exo- and endoglucanases (23.70 and 34.72 U/mg protein, respectively). The crude exo- and endoglucanases had optimum activities at pH 8.0, 70 °C and pH 7.0, 50 °C, respectively. The enzymes were stable over pH of 4.0–7.0 with relative residual activity above 60% after 1 h incubation. Exoglucanase activity was enhanced by Ca²⁺ and Cu²⁺ at 5 mM and Mg²⁺ at 10 mM. Endoglucanase activity was greatly enhanced in the presence of Mn²⁺, Ca²⁺, Mg²⁺, Cu²⁺ and Fe³⁺ at 5 and 10 mM. Activities of both enzymes were inhibited in the presence of Hg²⁺ at 5 and 10 mM. Results show that F. oxysporum possessed good cellulolytic enzyme system for efficient conversion of cellulose. Exhibited thermotolerance of exoglucanase with the striking tolerance of endoglucanase to metal ions demonstrate potentials of enzymes for biofuel industry.     

Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris

Bioconversion of cellulosic material into glucose needs cellulase enzymes. One of the most important organisms that produces cellulases is Trichoderma reesei, whose cellulose enzymes are probably the most widely used in the industry. However, these enzymes are not stable enough at high pH and temperatures. The optimized synthetic endoglucanase II gene with Pichia pastoris codon preferences was secretary expressed in P. pastoris. Recombinant enzyme characterization showed maximum activity at pH 4.8 and temperature 75 °C, and it demonstrated increasing thermal stability in high temperature. The enzyme maintained its activity in a wide pH range from 3.5 to 6.5. The optimization of fermentation medium was carried out in shaking flasks. Recombinant protein expression at optimum conditions (pH 7, temperature 25 °C, and 1 % methanol induction) for 72 h demonstrated 2,358.8 U/ml endoglucanase activity units. To our knowledge, this is the highest acidic thermophilic endoglucanase activity that is reported in crude intracellular medium in P. pastoris. We conclude that P. pastoris is an appropriate host for high-level expression of optimized endoglucanase gene with improved thermal stability.     

Influences of Media Compositions on Characteristics of Isolated Bacteria Exhibiting Lignocellulolytic Activities from Various Environmental Sites

Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.     

Improvement of Bioethanol Productivity of Immobilized Saccharomyces Bayanus with Using Sodium Alginate-Graft-Poly(N-Vinyl-2-Pyrrolidone) Matrix

In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84?gL^?1?h^?1 when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84?gL^?1?h^?1 while the glucose concentration increasing from 50 to 100?gL^?1. Due to the increase in percentage from 10 to 20?% of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68?gL^?1?h^?1. The cell immobilized NaAlg-g-PVP beads were protected 92?% of initial activity after six repeated fermentation.     

Siderophore-Mediated Iron Uptake Promotes Yeast–Bacterial Symbiosis

In the present study, siderophore produced by the marine yeast Aureobasidium pullulans was characterized as hydroxamate by chemical and bioassays. The hydroxamate assignment was supported by the appearance of peaks at 1,647.21?C1,625.99?cm^?1 and at 1,435.04?cm^?1 in the infrared spectrum. The purified siderophore exhibited specific growth-promoting activity under iron-limited conditions for siderophore auxotrophic probiotic bacteria. Cross-utilization of siderophore indicates a symbiotic relationship between the yeast A. pullulans and the selected probiotic bacterial strains. Statistical optimization of medium components for improved siderophore production in A. pullulans was depicted by response surface methodology. The shift in UV?CVis spectroscopy indicates the photoreactive property and subsequent oxidative cleavage of purified siderophore on exposure to sunlight.     

Extracellular Production of Novel Halotolerant, Thermostable, and Alkali-Stable Carboxymethyl Cellulase by Marine Bacterium Marinimicrobium sp. LS-A18

Cellulases which are active and stable under extreme conditions have attracted considerable attention because of their potential industrial applications. Marinimicrobium sp. LS-A18 showed high extracellular carboxymethylcellulase (CMCase) activity when grown on mineral salt medium containing carboxymethylcellulose as the sole carbon source. Maximum CMCase activity was obtained at 55°C and pH 7.0 in the absence of NaCl. Under the optimized fermentation conditions, the yield of CMCase was increased up to 2.5 U/ml, which was 3.1-fold higher than that before optimization. The enzyme retained 84?% of residual activity after incubation at 60°C for 1?h and more than 88?% of residual activity after incubation for 72?h in the presence of different pH (5?C11) and NaCl concentrations (0?C25?%, w/v), indicating it was halotolerant, thermostable and alkali-stable. These characteristics made the CMCase from Marinimicrobium sp. LS-A18 as a potentially novel biocatalyst in biotechnological and industrial applications.     

Production of Microbial Cellulose by a Bacterium Isolated from Fruit

This study presents the production of bacterial cellulose (BC) by a bacterium isolated from a rotten fruit and its process optimization. Here, isolation and screening of potent cellulose producers were carried out from different natural sources, viz., soil, rotten fruits, and vegetables and vinegar. A total of 200 bacterial isolates were obtained, which were screened for cellulose production using Hestrin?CSchramm medium. A novel and potent cellulose-producing bacterium was newly isolated from a rotten fruit and identified as Gluconacetobacter sp. F6 through 16S ribosomal DNA sequencing and morphological, cultural, and biochemical characteristics. After optimization of culture conditions, including pH, temperature, agitation, carbon/nitrogen sources, and inducers, the BC production was greatly increased from 0.52 to 4.5?g/l (8.65-fold increase). The optimal culture medium contained 1% (w/v) glucose, 1.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.27% (w/v) disodium hydrogen phosphate, 0.115% (w/v) citric acid, and 0.4% (w/v) ethanol. BC produced was analyzed for the presence of cellulose fibrils by epiflourescent microscopy using Calcofluor white stain and scanning electron microscopy and confirmed by NMR. There are very scanty reports about the optimization of BC production by bacteria isolated from rotten fruits.     

Alaninyl variants of the marine natural product halocyamine A and their antibacterial properties

In an effort to explore the antibacterial potential of the marine natural product halocyamine A, a series of analogues including desbromo and alanine-substituted variants were synthesised and evaluated for biological activity against a panel of Gram-positive and –negative bacteria. The analogues were synthesised by a combination of solid-phase peptide synthesis and ruthenium complex/ytterbium triflate catalysed hydroamidation chemistry. Single alanine substitutions ([Ala¹]-halocyamine A and [Ala²]-halocyamine A) gave only modest increases in activity towards Gram-positive bacteria, while di-alaninyl variants exhibited more potent activity with MIC values of 12.5–50?μM towards the Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis. A lipophilic trityl-protected intermediate of [Ala²]-halocyamine was the most active against the Gram-negative bacterium Escherichia coli.     

Stability and recovery ofPenicillium funiculosum cellulase under use conditions

The stability ofPenicillium funiculosum cellulase has been investigated under the conditions used for cellulose hydrolysis. Fifty five percent of filter paper activity (FPA) was inactivated on incubation at 50°C for 24 h, whereas there was no loss in endoglucanase and β-glucosidase activity. The addition of 2% polyethylene glycol (PEG) during incubation stabilized the FPA. The influence of pH during fermentation on the thermal stability of the enzyme is discussed. The recovery of enzymes after hydrolysis of bagasse at 50°C was between 8 and 14%. Under the optimal conditions of elution, the recovery of enzyme was 35% (1). Increasing the enzyme to the substrate ratio fivefold and presence of PEG during hydrolysis resulted in 80, 83, and 95% recovery of β-glucosidase, FPA, and endoglucanase activity, respectively. Index Entries: Stability; recovery of cellulase P.funiculosum.     

Xylanase Isozymes from the Newly Isolated Bacillus sp. CKBx1D and Optimization of Its Deinking Potentiality

Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480?U/ml) at 36?h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94?kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2?h of continuous shaking at constant temperature of 35?°C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.     

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.     

Enhancing the Cellulose-Degrading Activity of Cellulolytic Bacteria CTL-6 (Clostridium thermocellum) by Co-Culture with Non-cellulolytic Bacteria W2-10 (Geobacillus sp.)

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml^?1 and 45.1 μg ml^?1 (DNA content) up to 0.47 U ml^?1 and 112.2 μg ml^?1, respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml^?1. This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.     

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2 × 10⁶ mL⁻¹. Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.     

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties

Background: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. Methods: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. Results: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. Conclusions: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.     

Identification of Newly Zeaxanthin-Producing Bacteria Isolated from Sponges in the Gulf of Thailand and their Zeaxanthin Production

Sponge-associated bacteria have been found to produce a variety of bioactive compounds including natural pigments. Here, we report the molecular identification of zeaxanthin-producing sponge-associated bacteria isolated from sponges in the Gulf of Thailand and the effect of environmental factors on zeaxanthin production from a bacterium. Three colorful sponge-associated bacteria (CHOB06-6, KODA19-6, and MAKB08-4) were identified based on the 16S rDNA profile. The 16S rDNA sequence-based analyses revealed that CHOB 06-6 and MAKB 08-4 were the closest relatives to Sphingomonas phyllosphaerae FA2^T, and KODA19-6 was a relative of Shingomonas (Blastomonas) natatoria DSM 3183^T. After all bacteria were cultivated in a modified Zobell medium, S. natatoria KODA19-6 was found to produce the highest zeaxanthin at 0.62?mg/l. pH and temperature considerably affected its zeaxanthin production. Its optimal condition for zeaxanthin production was found at a pH of 7 and 30?°C. The bacterium had a maximum specific growth rate (?? _max) of 0.06?1/h with zeaxanthin productivity (Q _p) of 6.27???g/l·h. Therefore, this newly zeaxanthin-producing bacterium has a potential to produce natural zeaxanthin for the food, feed, pharmaceutical, and cosmetic industries.     

Screening, Gene Sequencing and Characterising of Lipase for Methanolysis of Crude Palm Oil

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K _M of 0.75 mM and a V _max of 0.33 mM?min^?1 on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.