Efficient production of a novel alkaline cold-active phospholipase C from Aspergillus oryzae by molecular chaperon co-expression for crude oil degumming.

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.

Effect of phenolic compounds from the leaves of Psidium guajava on the activity of three metabolism-related enzymes.

Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer); due to the numerous side effects of the latter. In vitro screening assays were conducted for Psidium guajava leaf methanolic extract against three metabolism-related enzymes; α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained weak and moderate multitarget inhibition against α-amylase, tyrosinase, and hyaluronidase, respectively; however, the leaf fractions exhibited stronger inhibitions for the three investigated enzymes. Fractionation of P. guajava leaf extract revealed that anthraquinones and ellagic acid are of the major active compounds with inhibitory activities for α-amylase, tyrosinase, and hyaluronidase. Kinetic studies showed that quinalizarin inhibition is competitive for both α-amylase and hyaluronidase, and ellagic acid inhibition for tyrosinase and hyaluronidase is competitive and un-competitive, respectively. The molecular docking studies of quinalizarin and ellagic acid with α-amylase, tyrosinase, and hyaluronidase showed high binding energies with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. guajava leaf fractions, quinalizarin and ellagic acid, have multitarget activities with potential therapeutic applications in many metabolic disorders.

Effect of phenolic compounds from the rind of Punica granatum on the activity of three metabolism-related enzymes.

Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer) due to the numerous side effects of the latter. In vitro screening assays were conducted for Punica granatum rind methanolic extract against three metabolism-related enzymes: α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained high multitarget inhibition with inhibition percentages 31.5 ± 1.3%, 75.9 ± 4.7%, and 68.5 ± 5.3% against α-amylase, tyrosinase, and hyaluronidase, respectively. Bioguided fractionation of P. granatum rind extract revealed that quercetin is the major active compound with inhibitory activities: 54.3 ± 2.7%, 94.2 ± 3.5%, and 90.9 ± 2.7% against α-amylase, tyrosinase, and hyaluronidase, respectively. Kinetic studies of enzymes showed that quercetin inhibition was noncompetitive, uncompetitive, and competitive for α-amylase, tyrosinase, and hyaluronidase, respectively. The molecular docking of quercetin with α-amylase and hyaluronidase showed high binding energy with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. granatum rind extract and quercetin have multitarget activities with potential therapeutic applications in many metabolic disorders.

Discovery of Novel High-Molecular Weight Oligosaccharides Containing N-Acetylhexosamine in Bovine Colostrum Whey Permeate Hydrolyzed with Aspergillus oryzae ß-Galactosidase.

Bovine milk oligosaccharides (BMOs) that resemble human milk oligosaccharides are found in whey permeate, indicating that dairy streams can be used as a potential source of bioactive oligosaccharides. Recovery of oligosaccharides from whey permeate is hindered by their low abundance and high concentration of lactose. In the present work, lactose in bovine colostrum whey permeate was hydrolyzed by Aspergillus oryzae ß-galactosidase to facilitate subsequent monosaccharide removal by membrane separation. Chromatographic separation coupled with high-resolution mass spectrometry revealed ß-galactosidase degradation of several ß-linkage-containing BMOs and production of novel oligosaccharides that ranged in size from 5 to 11 monosaccharide units containing several galactose repeating units and N-acetylhexosamine at their reducing ends. Optimization of BMO hydrolysis and separation methodology could generate high amounts of hetero-oligosaccharides for improved recovery of potentially biotherapeutic oligosaccharides.

Effect of Agave americana L. on the human, and Aspergillus oryzae S2 α-amylase inhibitions.

Among phenolic compounds, Agave americana L. extract contained puerarin (38.4%) and p-coumaric acid (12.29%) (pCa). From the Lineweaver-Burk plots, pCa and puerarin demonstrated a competitive and a non competitive inhibitions towards human α-amylase activity, respectively. PCa exhibited a higher human inhibitory activity with an IC50 of 98.8 µM which was about 2.3 times than acarbose. Puerarin (IC50 = 3.87 µM) and pCa (IC50 = 10.16 µM) also showed an excellent inhibition for Aspergillus oryzae S2 α-amylase activity. The inhibitions of the described biocatalysts compounds towards both amylases were significantly decreased when they were pre-incubated with starch. The binding modes of these compounds were evaluated in silico. The binding efficiency order of these molecules in terms of polar contact numbers for both enzymes was in agreement with the in vitro studies. These findings provided a rational reason to establish the isolated compounds capability as therapeutic target for hyperglycaemia modulation and antifungal therapy.

Measuring Bacterial Glycosyl Hydrolase Activity with a Soluble Capture Probe by Mass Spectrometry.

A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes.

The microbial production of fumaric acid by Rhizopus arrhizus NRRL 2582 has been evaluated using soybean cake from biodiesel production processes and very high polarity (VHP) sugar from sugarcane mills. Soybean cake was converted into a nutrient-rich hydrolysate via a two-stage bioprocess involving crude enzyme production via solid state fermentations (SSF) of either Aspergillus oryzae or R. arrhizus cultivated on soybean cake followed by enzymatic hydrolysis of soybean cake. The soybean cake hydrolysate produced using crude enzymes derived via SSF of R. arrhizus was supplemented with VHP sugar and evaluated using different initial free amino nitrogen (FAN) concentrations (100, 200, and 400 mg/L) in fed-batch cultures for fumaric acid production. The highest fumaric acid concentration (27.3 g/L) and yield (0.7 g/g of total consumed sugars) were achieved when the initial FAN concentration was 200 mg/L. The combination of VHP sugar with soybean cake hydrolysate derived from crude enzymes produced by SSF of A. oryzae at 200 mg/L initial FAN concentration led to the production of 40 g/L fumaric acid with a yield of 0.86 g/g of total consumed sugars. The utilization of sugarcane molasses led to low fumaric acid production by R. arrhizus, probably due to the presence of various minerals and phenolic compounds. The promising results achieved through the valorization of VHP sugar and soybean cake suggest that a focused study on molasses pretreatment could lead to enhanced fumaric acid production.

Purification and characterization of a protease-resistant phytase of Aspergillus oryzae SBS50 whose properties make it exceptionally useful as a feed supplement.

An extracellular phytase of Aspergillus oryzae SBS50 was purified to homogeneity using ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Purified phytase has a monomeric molecular mass of ∼80kDa exhibiting its optimal activity at pH 5.0 and 50°C with a T 1/2 of 300min at 50°C. Phytase of A. oryzae displayed broad substrate specificity with Vmax and Km values of 58.82µmol/ml/min and 1.14mM, respectively, for calcium phytate. Purity and homogeneity of the phytase was confirmed by high performance liquid chromatography and MALDI-TOF analysis revealed the identification of a peptide showing homology with acid phosphatase of Aspergillus oryzae RIB40. Among the inhibitors, 2,3-butanedione and sodium molybdate significantly inhibited the enzyme activity. Phytase of A. oryzae showed protease-resistance and was more stable during storage at 4°C and -20°C as compared to room temperature. Among all the feed samples, mustard oil cake was dephytinized more efficiently than other feed samples. These unique properties suggested that the phytase has the potential to be useful as an animal feed supplement.

Immobilization of Enzymes on PLGA Sub-Micrometer Particles by Crosslinked Layer-by-Layer Deposition.

Enzyme immobilization is of high interest for industrial applications. However, immobilization may compromise enzyme activity or stability due to the harsh conditions which have to be applied. The authors therefore present a new and improved crosslinked layer-by-layer (cLbL) approach. Two different model enzymes (acid phosphatase and ß-galactosidase) are immobilized under mild conditions on biocompatible, monodisperse, sub-micrometer poly(lactide-co-glycolide) (PLGA) particles. The resulting PLGA enzyme systems are characterized regarding their size, surface charge, enzyme activity, storage stability, reusability, and stability under various conditions such as changing pH and temperature. The developed and characterized cLbL protocol can be easily adapted to different enzymes. Potential future uses of the technology for biomedical applications are discussed. PLGA-enzyme particles are therefore injected into the blood circulation of zebrafish embryos in order to demonstrate the in vivo stability and activity of the designed system.

Effects of nonionic surfactants on pellet formation and the production of ß-fructofuranosidases from Aspergillus oryzae KB.

Aspergillus oryzae KB produces two ß-fructofuranosidases (F1 and F2). F1 has high transferring activity and produces fructooligosaccharides from sucrose. Mycelial growth pellets were altered by the addition of Tween 20, 40 and 80 (HLB=16.7, 15.6 and 15.0, respectively) in liquid medium cultures to form small spherical pellets. The particle size of the pellets decreased with the HLB value, which corresponds to an increase in surfactant hydrophobicity. Selective F1 production and pellet size were maximized using Tween 20. Adding polyoxyethylene oleyl ethers (POEs) with various degrees of polymerization (2, 7, 10, 20 and 50: HLB=7.7, 10.7, 14.7, 17.2 and 18.2, respectively) was investigated. A minimum mean particle size was obtained using a POE with DP=10, HLB=14.7. The POE surfactants had little effect on the selective production of F1. The formation of filamentous pellets depended on the surfactant HLB value, and F1 enzymes were produced most efficiently using Tween 20.

Identification of a novel Penicillium chrysogenum rhamnogalacturonan rhamnohydrolase and the first report of a rhamnogalacturonan rhamnohydrolase gene.

Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of ß-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.

Effect of Tea Catechins on Folate Analysis in Green Tea by Microbiological Assay.

Green tea is thought to be a primary source of folate in the Japanese diet, based on folate content analyzed by a microbiological assay. Green tea also contains high amount of catechins, in particular, epigallocatechin gallate (EGCg), which was demonstrated to be able to inhibit the digestive enzyme activities and microbial growth in the folate assay. In the present study, we examined whether tea catechins interfered with components of the folate assay for green tea. A marked inhibitory effect of EGCg on microbial growth was observed at an inhibitory concentration of higher than 10 µg/mL. Tea catechins without the galloyl moiety did not show an inhibitory effect. EGCg inhibited the activity of the three enzymes used for assay sample preparation at an inhibitory concentration of higher than 750 µg/mL for α-amylase, 1,000 µg/mL for protease, and 50 µg/mL for conjugase. However, with each step of the assay, the actual concentration of EGCg was decreased to below the inhibitory concentration of each analytical step. Lack of influence of EGCg on green tea folate assay was confirmed by an addition of folate standard in tea infusion. These results suggested that tea catechins have no practical impact on folate analysis in green tea, using the general microbiological assay.

Modeling lactose hydrolysis for efficiency and selectivity: Toward the preservation of sialyloligosaccharides in bovine colostrum whey permeate.

Enzymatic hydrolysis of lactose has been shown to improve the efficiency and selectivity of membrane-based separations toward the recovery of bioactive oligosaccharides. Achieving maximum lactose hydrolysis requires intrinsic process optimization for each specific substrate, but the effects of those processing conditions on the target oligosaccharides are not well understood. Response surface methodology was used to investigate the effects of pH (3.25-8.25), temperature (35-55°C), reaction time (6 to 58 min), and amount of enzyme (0.05-0.25%) on the efficiency of lactose hydrolysis by ß-galactosidase and on the preservation of biologically important sialyloligosaccharides (3'-siallylactose, 6'-siallylactose, and 6'-sialyl-N-acetyllactosamine) naturally present in bovine colostrum whey permeate. A central composite rotatable design was used. In general, ß-galactosidase activity was favored at pH values ranging from 3.25 to 5.75, with other operational parameters having a less pronounced effect. A pH of 4.5 allowed for the use of a shorter reaction time (19 min), lower temperature (40°C), and reduced amount of enzyme (0.1%), but complete hydrolysis at a higher pH (5.75) required greater values for these operational parameters. The total amount of sialyloligosaccharides was not significantly altered by the reaction parameters evaluated, suggesting specificity of ß-galactosidase from Aspergillus oryzae toward lactose as well as the stability of the oligosaccharides at pH, temperature, and reaction time evaluated.

Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

Water-soluble benzodiazepine prodrug/enzyme combinations for intranasal rescue therapies.

Benzodiazepines (BZDs), including diazepam (DZP) and midazolam (MDZ), are drugs of choice for rapid treatment of seizure emergencies. Current approved use of these drugs involves administration via either intravenous or rectal routes. The former requires trained medical personnel, while the latter is socially unacceptable for many patients and caregivers. In recent years, efforts have been made to formulate BZDs for nasal administration. Because of the low solubility of these molecules, organic vehicles have been used to solubilize the drugs in the nasal products under development. However, organic solvents are irritating, potentially resulting in injury to nasal tissue. Here we report preliminary studies supporting a strategy in which water-soluble BZD prodrugs and a suitable converting enzyme are coadministered in an aqueous vehicle. Diazepam and midazolam prodrugs were synthesized and were readily converted to their active forms by a protease from Aspergillus oryzae. Using a permeation assay based on monolayers of Madin-Darby canine kidney II-wild type cells, we found that enzymatically produced BZDs could be maintained at high degrees of supersaturation, enabling faster transport across the membrane than can be achieved using saturated solutions. This strategy not only obviates the need for organic solvents, but it also suggests more rapid absorption and earlier peak concentrations than can be otherwise achieved. This article is part of a Special Issue entitled "Status Epilepticus".

Selenium speciation in bay scallops by high performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection after complete enzymatic extraction.

Selenium (Se) species, Se-methyl-seleno-cysteine (MeSeCys), seleno-cystine (SeCys2), seleno-methionine (SeMet), selenite (SeO3(2-)) and selenate (SeO4(2-)), in the three main anatomical tissues of bay scallops (Argopecten irradians), the adductor muscle, the mantle and the visceral mass, were completely released by enzymatic hydrolysis and detected by high performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS). For the thorough hydrolysis of the proteins to free the Se species, bay scallop tissues were pre-treated (pre-hydrolyzed) with papain in a 1molL(-1) sodium bicarbonate solution containing 5mmolL(-1) sodium thiosulfate at 30-40°C for 24h, then hydrolyzed by the combination of Flavourzyme(®) 500 L, carboxypeptidase Y and trypsin (3+1+1) at 45°C, at a constant pH of 8.00 for 6h. Under the optimized conditions, the quantification limits of MeSeCys, SeCys2, SeMet, SeO3(2-) and SeO4(2-) were 0.69, 0.48, 0.93 0.53 and 1.22µgL(-1), respectively (equivalent to 0.14, 0.097, 0.19, 0.11 and 0.24µgg(-1) for real samples). The working curves in the concentration ranges of 2 to 500µgL(-1) were linear with all the RSD (n=5) smaller than 15% and regression coefficients greater than 0.999. The recoveries of the species for spiked samples at 4µgg(-1) (equivalent to 20µgL(-1) in the final hydrolyzates) levels all exceeded 90%. The developed method was validated by the determination of SeMet in SELM-1, a Se enriched yeast certified reference material (CRM). Selenate was the only absent species, whereas the other four species did exist in bay scallops.

High-efficiency removal of phytic acid in soy meal using two-stage temperature-induced Aspergillus oryzae solid-state fermentation.

BACKGROUND: Phytic acid of soy meal (SM) could influence protein and important mineral digestion of monogastric animals. Aspergillus oryzae (ATCC 9362) solid-state fermentation was applied to degrade phytic acid in SM. Two-stage temperature fermentation protocol was investigated to increase the degradation rate. The first stage was to maximize phytase production and the second stage was to realize the maximum enzymatic degradation. RESULTS: In the first stage, a combination of 41% moisture, a temperature of 37 °C and inoculum size of 1.7 mL in 5 g substrate (dry matter basis) favored maximum phytase production, yielding phytase activity of 58.7 U, optimized via central composite design. By the end of second-stage fermentation, 57% phytic acid was degraded from SM fermented at 50 °C, compared with 39% of that fermented at 37 °C. The nutritional profile of fermented SM was also studied. Oligosaccharides were totally removed after fermentation and 67% of total non-reducing polysaccharides were decreased. Protein content increased by 9.5%. CONCLUSION: Two-stage temperature protocol achieved better phytic acid degradation during A. oryzae solid state fermentation. The fermented SM has lower antinutritional factors (phytic acid, oligosaccharides and non-reducing polysaccharides) and higher nutritional value for animal feed.

Production optimization and expression of pectin releasing enzyme from Aspergillus oryzae PO.

Protopectinase is an enzyme that solubilizes protopectin forming highly polymerized soluble pectin. Protopectinase activity was detected from Aspergillus oryzae PO isolated from soil of persimmon orchard. Response surface methodology of Box-Behnken Design with three fermentation variables (temperature, NaNO3 and apple pomace concentration) was used to optimize protopectinase production of A. oryzae PO, and protopectinase activity was improved to 270.0 U/ml. Endo-polygalacturonase belonged to A-type PPase from A. oryzae PO was cloned and expressed in Pichia pastoris GS115. The endo-polygalacturonase expression was 0.418 mg/ml and the specific activity of purified recombinant endo-polygalacturonase was 7520 U/mg toward polygalacturonic acid. The optimal temperature and pH of recombinant endo-polygalacturonase were 45°C and 5.0, respectively. The recombinant endo-polygalacturonase activity was enhanced by the presence of Mg(2+), while Ca(2+), Ni(2+) Mn(2+), Cu(2+) and SDS strongly inhibited the enzyme activity. The apparent Km value and Vmax value were 5.59 mg/ml and 1.01 µmol/(minml), respectively.

Improved production of protease-resistant phytase by Aspergillus oryzae and its applicability in the hydrolysis of insoluble phytates.

Among three hundred isolates of filamentous fungi, Aspergillus oryzae SBS50 secreted higher phytase activity at pH 5.0, 35 °C and 200 rpm after 96 h of fermentation. Starch and beef extract supported the highest phytase production than other carbon and nitrogen sources. A nine-fold improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. Among surfactants tested, Tween 80 increased fungal growth and phytase production, which further resulted in 5.4-fold enhancement in phytase production. The phytase activity was not much affected by proteases treatment. The enzyme resulted in the efficient hydrolysis of insoluble phytate complexes (metal- and protein-phytates) in a time dependent manner. Furthermore, the hydrolysis of insoluble phytates was also supported by scanning electron microscopy. The enzyme, being resistant to trypsin and pepsin, and able to hydrolyze insoluble phytates, can find an application in the animal food/feed industry for improving nutritional quality and also in combating environmental phosphorus pollution and plant growth promotion.

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.