Improvement of a fed‐batch process for high level xylanase production by a Bacillus strain

In this paper, the improvement of a fed‐batch fermentation from the point of view of an industrial xylanase production process is described. The Bacillus strain chosen for this study is able to produce high quantities of a xylanase that is suitable to be used as bleach boost agent in chlorine‐free bleaching sequences of paper pulp. It was found that xylo‐oligosaccharides (hydrolysis products from xylan by xylanase action) were indispensable for induction of the enzyme synthesis, but that their presence in quantities of only 0.1 g dm^?3 xylose equivalents led to catabolite repression. A substrate‐limited fed‐batch process, that is the most adapted, was furthermore improved with regard to nutrient requirement of the microorganism, especially the nitrogen source. A process with constant supply of a culture medium containing xylan, peptone and mineral nitrogen was able to produce 20 240 nkat cm^?3 with a productivity of 910 nkat cm^?3 h^?1, which places the process among the best ever reported. © 2001 Society of Chemical Industry     

Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG6000/sodium citrate aqueous two-phase system

Aqueous two-phase system (ATPS) was applied for extraction bioconversion of xylan by xylanase from Trichoderma viride. Phase diagrams for poly (ethylene glycol) (PEG) and sodium citrate were determined at room temperature. The ATPS composed of 12.99% (w/w) PEG6000 and 12.09% (w/w) sodium citrate was favorable for partition of xylanase and used for extraction bioconversion of xylan. Batch hydrolysis demonstrated that higher concentrations of xylobiose and xylotriose were obtained in the PEG6000/sodium citrate ATPS compared to those in the aqueous system. These results present the potential feasibility of production of xylo-oligosaccharides by extraction bioconversion in ATPS.     

Wet oxidation pretreatment of lignocellulosic residues of sugarcane,rice, cassava and peanuts for ethanol production

Wet oxidation (WO) pretreatment of sugarcane bagasse, rice hulls, cassava stalks and peanut shells was investigated. WO was performed at 195 °C for 10 min, with 2 g kg^?1 of Na₂CO₃ and under either 3 or 12 bar of oxygen. Oxygen pressure and the type of raw material used had a major effect on the fractionation of the materials, formation of sugars and by‐products, and cellulose enzymatic convertibility. Cellulose content in the solid fraction increased after pretreatment of all materials, except rice hulls. The greatest increase, from 361 g kg^?1 to almost 600 g kg^?1, occurred for bagasse. The solubilisation of individual components was different for each material. Bagasse xylan was solubilised to a large extent, and 45.2% of it was recovered as xylose and xylo‐oligosaccharides in the liquid fraction. In the prehydrolysates of rice hulls around 40% of the original glucan was recovered as gluco‐oligosaccharides, due to hydrolysis of starch contained in grain remains. The formation of by‐products was modest for all the materials, but increased with increasing oxygen pressure. The highest yield of acetic acid (34–36 g kg^?1 of raw material) and furfural (0.7–1.8 g kg^?1) occurred for bagasse. The pretreatment enhanced the enzymatic convertibility of cellulose giving the best result (670.2 g kg^?1) for bagasse pretreated at the highest oxygen pressure. However, for the other materials the pretreatment conditions were not effective in achieving cellulose conversions above 450 g kg^?1. Some enzymatic conversion of xylan was observed. Copyright © 2007 Society of Chemical Industry     

Design of different bioreactor configurations: application to ligninolytic enzyme production in semi‐solid‐state cultivation

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) in laboratory‐scale bioreactors was studied. The cultivations were carried out in semi‐solid‐state conditions, employing corncob as carrier, which functioned both as a place of attachment and as a source of nutrients. Several bioreactor configurations were investigated in order to determine the most suitable one for ligninolytic enzyme production: a 1‐dm³‐static‐bed bioreactor, a 1‐dm³‐static‐bed bioreactor with air diffusers into the bed, a 0.5‐dm³‐static‐bed bioreactor with air diffusers into the bed and a tray bioreactor. Although the static‐bed configurations produced maximum individual lignin peroxidase (LiP) activities about 400 U dm⁻³ (1.0‐dm³ bioreactor) and about 700 U dm⁻³ (0.5‐dm³ bioreactor), manganese‐dependent peroxidase (MnP) was not detected throughout the cultures. Nevertheless, the tray configuration led to maximum individual MnP and LiP activities of about 200 U dm⁻³ and 300 U dm⁻³, respectively. Therefore, this configuration is the most adequate of the different bioreactor configurations tested in the present work, since the ligninolytic complex formed by MnP and LiP is more efficient for its application to bio‐processing systems. In addition, the results indicated the influence of the oxygen in ligninolytic enzyme production. © 2001 Society of Chemical Industry     

Poly(L‐lactide). X. Enhanced surface hydrophilicity and chain‐scission mechanisms of poly(L‐lactide) film in enzymatic,alkaline, and phosphate‐buffered solutions

Attempts were carried out to enhance the surface hydrophilicity of poly(L ‐lactide), that is, poly(L ‐lactic acid) (PLLA) film, utilizing enzymatic, alkaline, and autocatalytic hydrolyses in a proteinase K/Tris–HCL buffered solution system (37°C), in a 0.01N NaOH solution (37°C), and in a phosphate‐buffered solution (100°C), respectively. Moreover, its chain‐scission mechanisms in these different media were studied. The advancing contact‐angle (θ_a) value of the amorphous‐made PLLA film decreased monotonically with the hydrolysis time from 100° to 75° and 80° without a significant molecular weight decrease, when enzymatic and alkaline hydrolyses were continued for 60 min and 8 h, respectively. In contrast, a negligible change in the θ_a value was observed for the PLLA films even after the autocatalytic hydrolysis was continured for 16 h, when their bulk M_n decreased from 1.2 × 10⁵ to 2.2 × 10⁴ g mol^?1 or the number of hydrophilic terminal groups per unit weight increased from 1.7 × 10^?5 to 9.1 × 10^?5 mol g^?1. These findings, together with the result of gravimetry, revealed that the enzymatic and alkaline hydrolyses are powerful enough to enhance the practical surface hydrophilicity of the PLLA films because of their surface‐erosion mechanisms and that its practical surface hydrophilicity is controllable by varying the hydrolysis time. Moreover, autocatalytic hydrolysis is inappropriate to enhance the surface hydrophilicity, because of its bulk‐erosion mechanism. Alkaline hydrolysis is the best to enhance the hydrophilicity of the PLLA films without hydrolysis of the film cores, while the enzymatic hydrolysis is appropriate and inappropriate to enhance the surface hydrophilicity of bulky and thin PLLA materials, respectively, because a significant weight loss occurs before saturation of θ_a value. The changes in the weight loss and θ_a values during hydrolysis showed that exo chain scission as well as endo chain scission occurs in the presence of proteinase K, while in the alkaline and phosphate‐buffered solutions, hydrolysis proceeds via endo chain scission. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 1628–1633, 2003     

Modification of dextran through the grafting of N‐vinyl‐2‐pyrrolidone and studies of physicochemical phenomena in terms of metal‐ion uptake,swelling capacity,and flocculation

The optimum conditions for grafting N‐vinyl‐2‐pyrrolidone onto dextran initiated by a peroxydiphosphate/thiourea redox system were determined through the variation of the concentrations of N‐vinyl‐2‐pyrrolidone, hydrogen ion, potassium peroxydiphosphate, thiourea, and dextran along with the time and temperature. The grafting ratio increased as the concentration of N‐vinyl‐2‐pyrrolidone increased and reached the maximum value at 24 × 10^?2 mol/dm³. Similarly, when the concentration of hydrogen ion increased, the grafting parameters increased from 3 × 10^?3 to 5 × 10^?3 mol/dm³ and attained the maximum value at 5 × 10^?3 mol/dm³. The grafting ratio, add‐on, and efficiency increased continuously with the concentration of peroxydiphosphate increasing from 0.8 × 10^?2 to 2.4 × 10^?2 mol/dm³. When the concentration of thiourea increased from 0.4 × 10^?2 to 2.0 × 10^?2 mol/dm³, the grafting ratio attained the maximum value at 1.2 × 10^?2 mol/dm³. The grafting parameters decreased continuously as the concentration of dextran increased from 0.6 to 1.4 g/dm³. An attempt was made to study some physicochemical properties in terms of metal‐ion sorption, swelling, and flocculation. Dextran‐g‐N‐vinyl‐2‐pyrrolidone was characterized with infrared spectroscopy and thermogravimetric analysis. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

新型功能性食品基料──木低聚糖的研制

综述了木低聚糖的研究进展。迄今为止,制备木低聚糖的方法主要有以下四种：（1）酸水解法;（2）热水抽提法（包括蒸汽爆破法）;（3）生物酶降解法,包括间歇和连续两种生产方式;（4）微波降解法。     

D‐Lactic acid production from waste cardboard

The effects caused by alkaline treatment on the susceptibility of waste cardboard to enzymatic hydrolysis have been studied. Optimised conditions leading to extensive saccharification of both cellulose (870 g kg^?1 conversion) and hemicelluloses (845 g kg^?1 conversion) were identified. Samples treated under selected operational conditions were employed for producing D ‐lactic acid by simultaneous saccharification and fermentation (SSF) in media containing cellulases, β‐glucosidase and Lactobacillus coryniformis ssp torquens cells. SSF fed‐batch experiments led to D ‐lactic acid concentrations up to 23.4 g dm^?3 at a product yield of 514 g lactic acid kg^?1 of potential glucose and a volumetric productivity of 0.48 g dm^?3 h^?1. Copyright © 2004 Society of Chemical Industry     

Organosolv delignification of white‐ and brown‐rotted Eucalyptus grandis hardwood

Sound (undecayed control) and fungally‐pretreated wood samples were submitted to organosolv delignification. The cooking liquor used was methanol/water (78:22 v/v) containing CaCl₂ and MgSO₄ each at a concentration of 25 mmol dm⁻³. The cooking process was performed at 180 °C for reaction times varying from 5 to 100 min. Despite some differences in the lignin removal pattern, pseudo‐first order kinetic models permitted a prediction of delignification rate constants for all experiments. All biodegraded samples provided higher delignification rate constants than the undecayed control (2.0 × 10⁻²min⁻¹ for the undecayed control and, for example, 14.2 × 10⁻²min⁻¹ for the sample decayed by Trametes versicolor for 2.5 months). Biodegraded samples also presented significantly increased xylan removal rates. The type of biodegradation affected the behavior of wood samples under organosolv pulping. The highest delignification and xylan removal rate constants were observed in the sample decayed by T versicolor for 2.5 months (17% weight loss). However, high delignification and xylan removal rate constants were also observed in the sample decayed by Punctularia artropurpurascens for only 0.5 months (1.2% weight loss). Data obtained from a single fungal species pretreatment or data from all fungal pretreatments indicated that there is no clear correlation between the delignification constants and the wood weight or component losses. This lack of correlation suggested that the structure of residual polymers in decayed wood affects the delignification process in the organosolv pulping more than the removal extent of each individual component. © 2000 Society of Chemical Industry     

Enhanced ethanol production from enzymatically treated steam‐exploded rice straw using extractive fermentation

Alcohol fermentation of an enzymatic hydrolyzate of exploded rice straw was studied experimentally. Rice straw was treated under variable conditions, such as steam pressure and steaming time. The exploded rice straw was separated into water‐soluble material, methanol‐soluble lignin, Klason lignin, and a mixture of cellulose and a low molecular weight substance. The effects of steam explosion on the characteristics of the exploded rice straw were clarified from the point of view of the amounts of extractive components. Steam explosion was found to be effective for the delignification of rice straw and for increasing its susceptibility to enzyme hydrolysis and alcohol fermentation. The polysaccharides (cellulose and hemicellulose) in the rice straw treated at a steam pressure of 3.5 MPa with a steaming time of 2 min were hydrolyzed almost completely into monosaccharides, (ie glucose and xylose) by a mixture of Trichoderma viride cellulase (Meicelase) and Aspergillus aculeatus cellulase (Acucelase). The enzymatic hydrolyzate of exploded rice straw was converted into ethanol efficiently by Pichia stipitis and the ethanol yield from sugar was about 86%(w/w) of the theoretical value. The ethanol concentration in a membrane bioreactor coupled with a pervaporation system reached 50 gdm^?3 and was about five times higher than that in the culture broth. The energy efficiency (ratio of combustion energy of ethanol produced to energy for steam explosion) reached a maximum value at a pressure of 3.5 MPa for 2 min. © 2001 Society of Chemical Industry     

Production of Xylooligosaccharides from Waste Xylan,Obtained from Viscose Fiber Processing,by Selective Hydrolysis Using Concentrated Acetic Acid

Xylooligosaccharides (XOS) were prepared by selective hydrolysis of the waste xylan, obtained from viscose fiber plants, using concentrated acetic acid. The influences of acetic acid concentration, reaction temperature, and reaction time on the yield and component profiles of XOS product were investigated. These results were further ascertained by designing orthogonal experiments. The hydrolysis of xylan residue was selective, since mainly xylooligosaccharide components were formed, with hardly any impure ingredients, except for a small amount of xylose and traces of furfural, in the hydrolyzed product. Reaction temperature was found to be the most significant factor, influencing the XOS yield. Accurate HPAEC–PAD analysis of component profiles indicated that a maximum XOS yield of 45.86% was achieved on hydrolysis of 33.77 g/L waste xylan using 20% acetic acid for 20 min at 140°C. The main XOS components obtained were xylobiose, xylotriose, and xylotetraose, which were more than xylopentaose and xylohexaose.     

Reaction mechanisms and kinetics of xylo‐oligosaccharide hydrolysis by dicarboxylic acids

Hydrothermal pretreatment of lignocellulosic materials generates a liquid stream rich in pentose sugar oligomers. Cost‐effective hydrolysis and utilization of these soluble sugar oligomers is an integral process of biofuel production. We report integrated rate equations for hydrolysis of xylo‐oligomers derived from pretreated hardwood by dicarboxylic maleic and oxalic acids. The highest xylose yield observed with dicarboxylic acids was 96%, and compared to sulfuric acid, was 5–15% higher with less xylose degradation. Dicarboxylic acids showed an inverse correlation between xylose degradation rates and acid loadings unlike sulfuric acid for which less acid results in less xylose degradation to aldehydes and humic substances. A combination of high acid and low‐temperature leads to xylose yield improvement. Hydrolysis time course data at three different acid concentrations and three temperatures between 140 and 180°C were used to develop a reaction model for the hydrolysis of xylo‐oligosaccharides to xylose by dicarboxylic acids. © 2012 American Institute of Chemical Engineers AIChE J, 59: 188–199, 2013     

Production of delta‐endotoxin by Bacillus thuringiensis subsp kurstaki and overcoming of catabolite repression by using highly concentrated gruel and fish meal media in 2‐ and 20‐dm3 fermenters

Delta‐endotoxin production by a strain of Bacillus thuringiensis subsp kurstaki exhibiting larvicidal toxicity towards lepidoptera was investigated in 2‐ and 20‐dm³ fermenters, using gruel‐ and fish meal‐based media. The results show clearly that in such complex media, aeration plays an important role in bioinsecticide production. Optimal aeration led to improvement of delta‐endotoxin concentrations with decreases of final spore count and proteolytic activity. Moreover, in order to use high gruel concentrations, a fermenter configuration with an efficient aeration system should be used. In a 20‐dm³ Biolafite fermenter, 59 g dm^?3 or 75 g dm^?3 gruel was used to produce bioinsecticides with a significant reduction of carbon catabolite repression of delta‐endotoxin synthesis. This result is very interesting in order to produce high final delta‐endotoxin concentrations in the culture broth. It was also concluded, by considering the key role of oxidative pathways in delta‐endotoxin synthesis, that oxygen supply must be adequate for bioinsecticide production at high substrate concentrations. Moreover, the role of sodium chloride in improving delta‐endotoxin production is dependent not only on protease synthesis and its effect on crystal stability, but also on the aeration level of the production medium. © 2002 Society of Chemical Industry     

Production of fructose and ethanol from media with high sucrose concentrations by a mutant of Saccharomyces cerevisiae

The production of enriched fructose syrups and ethanol from a synthetic medium with high sucrose concentrations was studied in a batch process using Saccharomyces cerevisiae ATCC 36858. The results showed that the fructose yield was above 92% of theoretical values in synthetic media with sucrose concentrations between 180 g dm^?3 and 726 g dm^?3. Ethanol yield was about 82% in media with sucrose concentrations up to 451 g dm^?3. A product containing 178 g dm^?3 fructose, which represents 97% of the total sugar content, and 79 g dm^?3 ethanol was obtained using a medium with 360 g dm^?3 sucrose. The fructose fraction in the carbohydrates content in the produced syrups decreased with increases in the initial sucrose concentration. In a medium with initial sucrose concentration of 574 g dm^?3, the fructose content in the produced broth was 59% of the total carbohydrates. Glycerol and fructo‐oligosaccharides were also produced in this process. The produced fructo‐oligosaccharides started to be consumed when the concentration of sucrose in the media was less than 30% of its initial value. Complete hydrolysis of these sugars was noticed in media with sucrose concentrations below 451 g dm^?3. These findings will be useful in the production of ethanol and high fructose syrups using sucrose‐based raw materials with high concentrations of this carbohydrate. © 2001 Society of Chemical Industry     

High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his‐tagged rhamnulose‐1‐phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm^?3 and 288 AU dm^?3 h^?1 respectively) compared with Luria–Bertoni (LB) medium (2292 AU dm^? 3 and 255 AU dm^?3 h^?1). The minimum quantity of (isopropyl‐β‐D ‐thiogalactoside) IPTG for optimal induction was estimated in 18–20 µmol IPTG gDCW^?1. The highest volumetric production of RhuA (8333 AU dm^?3) was obtained when IPTG was added in the late log‐phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37 °C. An effective two‐step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications. Copyright © 2003 Society of Chemical Industry     

Production of fructooligosaccharides by β‐fructofuranosidase from Aspergillus sp 27H

β‐fructofuranosidase (EC 3.2.1.26) from Aspergillus sp 27H isolated from soil was investigated for production of fructooligosaccharides (FOS) using whole cells. It possesses hydrolytic and transfructosylating activities that can be altered by modifying the reaction conditions. The optimal conditions for the transfructosylating activity occur in the pH range 5.5–6.0 and at 60 °C, while hydrolytic activity was highest at pH 4.0 and 55 °C. At low sucrose concentration (10 g dm^?3) there was rapid conversion of sucrose to glucose and fructose and very low concentrations of FOS were obtained. However, at sucrose concentrations higher than 216 g dm^?3 the concentrations of hydrolysis products were reduced. Under the following conditions: pH 5.5, temperature 40 °C, sucrose concentration 615 g dm^?3 and enzyme concentration 20β‐fructofuranosidase units g^?1 of sucrose, the FOS concentration reached a maximum value of 376 g dm^?3 (234 g dm^?3 1‐kestose and 142 g dm^?3 nystose) and the proportion of FOS in the solids in the reaction mixture was 600–620 g kg^?1 at 6 h. These results suggest that β‐fructofuranosidase from Aspergillus sp 27H could be an appropriate enzyme for the commercial production of FOS. Copyright © 2004 Society of Chemical Industry     

The effect of an enzymatic pretreatment on the hydrolysis and size reduction of fat particles in slaughterhouse wastewater

The effect of an enzymatic pretreatment, Pancreatic Lipase 250 (PL‐250), on the hydrolysis and size reduction of fat particles in slaughterhouse wastewater was characterised for enzyme doses ranging from 125 to 1000 mg dm^?3 and initial particle sizes (D_in) varying between 53 and 383 µm. Treatment with PL‐250 significantly reduced the size of pork fat particles in slaughterhouse wastewater. Particle size reduction increased with D_in, possibly due to the more filamentous and plate‐like configuration of the larger fat particles, which could be easily broken at weak points. The smaller particles were observed to be denser and more spherical. Size reduction also increased with enzyme concentration, but the benefit of adding more enzyme diminished greatly as enzyme dose was increased. The maximum long‐chain fatty acid (LCFA) concentration in filtered samples was detected after 4–7 h of treatment and ranged from 8.2 to 34.9 mg dm^?3. The linear rate of LCFA released in solution during enzymatic pretreatment ranged from 39.4 to 169.9 mg dm^?3 d^?1, and increased with enzyme concentration up to 500 mg dm^?3. At a PL‐250 concentration of 1000 mg dm^?3, the LCFA release rate decreased, maybe due to excessive layering of adsorbed enzyme on the fat particles or increased degradation of released LCFAs. The pretreatment appeared to be more efficient with beef than pork fat particles. However, the effect of an enzymatic pretreatment on a downstream anaerobic treatment of slaughterhouse wastewater containing fat particles remains to be tested. © 2001 Society of Chemical Industry     

Anaerobic biodegradation of phenol in sulfide‐rich media

In the refinery industry, the washing processes of middle‐distillates using caustic solutions generate phenol‐ and sulfide‐containing waste streams. The spent caustic liquors generated contain phenols at concentrations higher than 60 g dm^?3(638.3 mmol dm^?3). For sulfur compounds, the average sulfide concentration was 48 g dm^?3(1500 mmol dm^?3) in these streams. The goal of this study was to evaluate the specific impact of phenol and sulfide concentrations towards the phenol‐biodegradation activity of a phenol‐acclimated anaerobic granular sludge. An inhibition model was used to calculate the phenol and sulfide inhibitory concentrations that completely stopped the phenol‐biodegradation activity (IC100). A maximum phenol‐biodegradation activity of 83 µmol g^?1 VSS h^?1 was assessed and the IC100 values were 21.8 mmol dm^?3 and 13.4 mmol dm^?3 for phenol and sulfide respectively. The limitation of the phenol biodegradation flow by phenol inhibition seemed to be related to the more important sensitivity of phenol‐degrading bacteria. The up‐flow anaerobic sludge bed reactor operating in a non‐phenol‐dependent inhibition condition did not present any sensitivity to sulfide concentrations below 9.6 mmol dm^?3. At this residual concentration, the pH and bisulfide ions' concentration might be responsible for the general collapsing of the reactor activity. Copyright © 2004 Society of Chemical Industry     

One pot synthesis of xanthan gum‐g‐N‐vinyl‐2‐pyrrolidone and study of their metal ion sorption behavior and water swelling property

In this article, graft copolymerization of N‐vinyl‐2‐pyrrolidone onto xanthan gum initiated by potassium peroxydiphosphate/Ag⁺ system in an aqueous medium has been studied under oxygen free nitrogen atmosphere. Grafting ratio, grafting efficiency, and add on increase on increasing the concentration of potassium peroxydiphosphate (2.0 × 10^?3 to 12 × 10^?3 mol dm^?3), Ag⁺(0.4 × 10^?3 to 2.8 × 10^?3 mol dm^?3), and hydrogen ion concentration from 2 × 10^?3 to 14.0 × 10^?3 mol dm^?3. Maximum grafting has been obtained when xanthan gum and monomer concentration were 0.4 g dm^?3 and 16 × 10^?2 mol dm^?3, respectively, at 35°C and 120 min. Water swelling capacity, swelling ratio, metal ion uptake, and metal retention capacity have also been studied, and it has been found that graft copolymer shows enhancement in these properties than pure xanthan gum. The graft copolymer has been characterized by FTIR and thermal analysis. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry