Physicochemical, functional and structural properties of vicilin-rich protein isolates from three Phaseolus legumes: Effect of heat treatment

The physicochemical, functional and structural properties of vicilin-rich protein isolates from kidney, red and mung beans (KPI, RPI and MPI) were investigated, and the influences of heating (at 95 °C for 30 min) were also evaluated and compared. In the untreated samples, the thermal stability, free SH contents and surface hydrophobicity were different. The differences seemed to be closely related to the differences in extent of aggregation and/or tertiary and secondary conformational structures among proteins. The heating resulted in extensive denaturation of the protein, significant decreases in free SH groups and increases in surface hydrophobicity, but to a varying extent, depending on the type of protein isolates. The protein solubility and emulsifying activities were significantly improved by the heating. The tertiary and secondary structures of these proteins were also to a various extent affected. The conformational structures of proteins in RPI were most flexible, and susceptible to the heating, while the proteins in KPI were most heat-stable in structures. The results clearly indicated close relationships between functional properties of these vicilin-rich protein isolates and their conformational structures.     

Thermal denaturation and gelation of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The thermal denaturation and gelation properties of vicilin-rich protein isolates from red bean, red kidney bean and mung beans (further denoted as RPI, KPI and MPI) were investigated by differential scanning calorimetry (DSC) and dynamic oscillatory measurements. The relation between the properties of these proteins to their free sulphydryl (SH)/disulfide bond (SS) contents was also evaluated. DSC analyses showed that many DSC characteristics of major endothermic peak (corresponding to vicilin component), including its denaturation temperature (T_d), enthalpy changes (ΔH) and width at half-peak height (ΔT_1/2), significantly varied with the type of protein isolates. Furthermore, the heat-induced gelation, including onset of gelation and the development of mechanical moduli, was also dependent on the type of protein isolates. The thermal denaturation of these proteins was nearly unaffected by the presence of reducing agent dithiothreitol (DTT), while the presence of DTT weakened the gel formation. The T_d of vicilin components and the mechanical moduli of corresponding formed gels were positively related to their SS contents. Additionally, the formed gels were thermo-irreversible, and heat pretreatment (carried out at temperatures higher than or close to the T_d of these vicilins) could improve the gel network formation. These results confirm that vicilin-rich protein isolates from various legumes show different patterns of thermal denaturation and gelation, and these properties are to a great extent related to their SH and/or SS contents.     

Thermal aggregation,amino acid composition and in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The heat-induced aggregation and the in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes, including kidney bean, red bean and mung bean were investigated and compared, and their amino acid composition and free sulfhydryl (SH) group contents also evaluated. The results showed that the extent in the heat-induced aggregation varied with the type of the protein isolates, and the formation of new disulphide bonds (at the expense of free SH contents) was involved in the formation of the aggregates. The protein isolates with higher levels of hydrophobic and uncharged polar amino acids, and lower basic amino acid contents exhibited lower extent of their heat-induced aggregation. The in vitro pepsin plus trypsin digestibility was different for various native protein isolates. The digestibility was to a varying extent affected by the heat treatment. The influences of heating on the digestibility of these proteins were closely related to the extent of their heat-induced aggregation. The results suggest that the improvement of nutritional property of those vicilin-rich protein isolates by heat treatment is largely dependent upon their amino acid composition as well as the extent of heat-induced aggregation.     

Microencapsulation properties of protein isolates from three selected Phaseolus legumes in comparison with soy protein isolate

The spray-drying microencapsulation properties of protein isolates from three selected Phaseolus legumes (kidney, red and mung beans; KPI, RPI and MPI), at a specific concentration (6 g/100 mL) and oil/protein ratio (1:1, w/w) were compared with soy protein isolate (SPI). The oil retention efficiency (RE), redispersion and dissolution behavior, as well as microstructure of the spray-dried powders were characterized. The influence of storage at 75% relative humidity for 7 days on these characteristics was also evaluated. The results indicated that the microencapsulation properties (except RE in the KPI case) of the three protein isolates were considerably poorer than SPI, though their emulsifying ability was even superior. The microencapsulating properties of these protein isolates were largely associated with their interfacial properties, especially the interfacial protein concentration. Among the three protein isolates, the spray-dried powders with KPI exhibited highest RE but least redispersion and/or dissolution behavior. The storage resulted in a severe loss of RE and ability to be redispersed and/or dissoluted, with much higher extent observed for the KPI powder. These results suggest that appropriate modifications, especially in interfacial properties, should be conducted on these proteins to warrant their application as wall materials in spray-drying microencapsulation.     

Effect of heat treatment of film-forming solution on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Effects of heat treatment at different temperatures (40–90 °C) of film-forming solution (FFS) containing 3% gelatin from cuttlefish (Sepia pharaonis) ventral skin and 25% glycerol (based on protein) on properties and molecular characteristics of resulting films were investigated. The film prepared from FFS heated at 60 and 70 °C showed the highest tensile strength (TS) with the highest melting transition temperature (T_max) (p < 0.05). Nevertheless, film from FFS heated at 90 °C had the highest elongation at break (EAB) with the highest glass transition temperature (T_g) (p < 0.05). With increasing heating temperatures, water vapor permeability (WVP) of films decreased (p < 0.05), but no differences in L*-value and transparency value were observed (p > 0.05). Based on FTIR spectra, the lower formation of hydrogen bonding was found in film prepared from FFS with heat treatment. Electrophoretic study revealed that degradation of gelatin was more pronounced in FFS and resulting film when heat treatment was conducted at temperature above 70 °C. Thus, heat treatment of FFS directly affected the properties of resulting films.     

Heat-induced modifications in the functional and structural properties of vicilin-rich protein isolate from kidney (Phaseolus vulgaris L.) bean

Heat-induced changes in the physico-chemical (and/or functional) and structural properties of protein isolate from kidney beans (KPI) were characterised. The extent of protein denaturation, free sulphydryl contents, surface hydrophobicity, as well as structural characteristics of the proteins were evaluated. Analyses of size-exclusion chromatography combined with laser scattering showed that the heating at 95 °C led to transformation of 7S-form vicilin to its 11S-form, and even higher molar mass (MW) oligomers or polymers. Moderate heating (for 15–30 min) significantly improved protein solubility, emulsifying and foaming activities (at neutral pH), whilst extensive heating (for 60–120 min) on the contrary decreased these properties. Spectral analyses of fluorescence and/or Raman spectroscopy showed that tertiary and secondary conformations of protein in KPI were remarkably affected to a varying extent by the heating. The results suggested a close relationship between functional properties of the vicilin from kidney bean and its conformational characteristics.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.     

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5 °C for 15 h), (ii) defrosting at room temperature (25 °C for 12 h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25 °C, microwave defrosting, defrosting at 5 °C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.     

Barrier property and penetration traces in packaging films against Plodia interpunctella (Hübner) larvae and Tribolium castaneum (Herbst) adults

The barrier property of different types of plastic film (with or without pinholes) against two insects, Plodia interpunctella (Hübner) larvae and Tribolium castaneum (Herbst) adults, and the morphology of damage produced in these insects were investigated. Using a penetration apparatus, four types of plastic films varying in thickness were used for insect-penetration tests: casted polypropylene, 20 μm and 25 μm (CPP20 and CPP25); oriented polypropylene, 20 μm and 30 μm (OPP20 and OPP30); linear low-density polyethylene, 40 μm and 50 μm (LLDPE40 and LLDPE50); and polyethylene terephthalate 12 μm and 16 μm (PET12 and PET16). After being fixed and tested in the penetration apparatus, each film was cut into a disc shape and 10 holes (200 μm diameter) were made by a pin. The shape of film damage and the mouthparts of insects were observed using scanning electronic microscopy. Plodia interpunctella larvae could penetrate all films with pinholes, while T. castaneum adults were unable to penetrate any of the films tested, even those with pinholes. The penetration-percentages by P. interpunctella larvae were 38% (LLDPE40), 3% (LLDPE), 53% (CPP20), 37% (CPP25), 63% (OPP20), 43% (OPP30), 83% (PET12) and 63% (PET16). The elongation value, tensile strength and thickness of film were important factors in the penetration test. LLDPE, which has the highest elongation value and the lowest tensile strength value, was the film that best protected against insect penetration. In CPP and LLDPE films, there were many scratches and tears around the holes. In comparison, much less damage was observed around the holes in OPP and PET films. By observing the mouthparts of insects, it was determined that P. interpunctella larvae had sharper mandibles than those of T. castaneum adults.     

Development of a novel bioactive packaging based on the incorporation of Lactobacillus sakei into sodium-caseinate films for controlling Listeria monocytogenes in foods

A novel packaging technology was developed based on the incorporation of Lactobacillus sakei cells into sodium-caseinate (SC) edible films. Incorporation was based either on direct addition of the cells in the film forming solution used for casting or by surface spraying of the culture on the preformed film, resulting in a population density of 10⁶ cfu/cm². Addition of sorbitol in the film matrix increased the viability of the cells, greater than 90%, upon storage under both refrigeration and ambient temperature conditions for 30 days. Incorporation of the viable protective culture did not affect the mechanical properties and the physico-chemical properties of the film. Application of the films to both laboratory medium (agar) and a food model system (fresh beef) inoculated with Listeria monocytogenes resulted in a rapid growth of L. sakei immobilized in the film following contact with the wet medium or the food surface and a significant inhibition of the pathogen growth compared to the control samples under both constant and dynamic storage temperature protocols. The present study indicated that biopolymer-based antimicrobial films containing cells of a protective culture can be used as an effective packaging technology for improving food safety.     

Functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate: Effect of high-pressure treatment

The effects of high-pressure (HP) treatment at 200–600 MPa, prior to freeze-drying, on some functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. Surface hydrophobicity and free sulfhydryl (SH) and disulfide bond (SS) contents were also evaluated. HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in fluorescence strength and SS formation from SH groups, and decrease in denaturation enthalpy change. The protein solubility of KPI was significantly improved at pressures of 400 MPa or higher, possibly due to formation of soluble aggregate from insoluble precipitate. HP treatment at 200 and 400 MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI was significantly decreased at 600 MPa (relative to untreated KPI). The thermal stability of the vicilin component was not affected by HP treatment. Additionally, in vitro trypsin digestibility of KPI was decreased only at a pressure above 200 MPa and for long incubation time (e.g., 120 min). The data suggest that some physiochemical and functional properties of vicilin-rich kidney proteins can be improved by means of high-pressure treatment.     

The effects of ultrasonic/microwave assisted treatment on the properties of soy protein isolate/titanium dioxide films

Soy protein isolate (SPI, 5.0 g/100 mL,) films embedded with nano-TiO₂ (0, 0.5, 1.0, 1.5 and 2.0 g/100 mL) were prepared by solution casting and modified by ultrasonic/microwave assisted treatment (UMAT). The effects of nano-TiO₂ content and UMAT time on the films' physical properties and structure were investigated. Incorporation of nano-TiO₂ significantly enhanced films' mechanical properties and barrier properties, because of the intermolecular force between nano-TiO₂ and SPI. UMAT time ≤20 min obviously improved films' tensile strength values (15.77 MPa, 245% higher than the control), and reduced water vapor permeability (1.8457 × 10⁻¹¹ g cm⁻¹ s⁻¹ Pa⁻¹, 72.11% lower than the control) and oxygen permeability values (0.8897 × 10⁻⁵ cm³ m⁻² d⁻¹ Pa⁻¹, 57.66% lower than the control). SEM images also revealed a more compact and dense structure of films when UMAT time ≤20 min. Films' water adsorption properties were evaluated. GAB and Henderson models exhibited the best to fit experimental data, thus it was predicted that films (1.5 g nano-TiO₂/100 ml) could be stable at low moisture content (0.27 kg of water/kg dry mass).     

Angiotensin-I converting enzyme inhibitory and antioxidant activities of protein hydrolysates from Phaseolus lunatus and Phaseolus vulgaris seeds

Phaseolus lunatus and Phaseolus vulgaris protein concentrates were hydrolyzed with the enzymes Alcalase^® and Flavourzyme^® at different reaction times, and the angiotensin-I converting enzyme (ACE-I) inhibitory activity, antioxidant properties and amino acid composition measured in the hydrolysates. With Alcalase^®, the highest degree of hydrolysis (DH) in P. lunatus was 37.94% at 45 min, and in P. vulgaris was 49.48% at 30 min. With Flavourzyme^®, the highest DH's were 22.03% and 26.05%, respectively, both at 90 min. ACE-I inhibitory activity in the Alcalase^® hydrolysates was IC₅₀ = 0.056 mg mL⁻¹ for P. lunatus at 90 min, and IC₅₀ = 0.061 mg mL⁻¹ for P. vulgaris at 60 min. In the Flavourzyme^® hydrolysates this activity was IC₅₀ = 0.0069 mg mL⁻¹ for P. lunatus at 90 min and IC₅₀ = 0.127 mg mL⁻¹ for P. vulgaris at 45 min. In SDS-PAGE, the hydrolysates exhibited low molecular weight bands. Antioxidant activity was 11.55 mmol L⁻¹ TEAC mg⁻¹ protein for P. lunatus with Flavourzyme^® at 90 min and 10.09 mmol L⁻¹ TEAC mg⁻¹ protein for P. vulgaris with Alcalase^® at 60 min. Amino acid composition exhibited high amino acid hydrophobic residues content.     

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.     

Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis

The global regulatory gene, afsR2, from Streptomyces lividans was previously reported to highly stimulate two structurally unrelated antibiotics, actinorhodin and undecylprodigiosin, in both S. lividans and its close relative S. coelicolor. Production of eight avermectin components was also improved in S. avermitilis: the use of wild-type S. avermitilis and its high-producing mutant, transformed by introduction of multiple copies of afsR2, increased the total avermectin productions by 2.3-fold and 1.5-fold, respectively.     

Heat-adaptation induced thermotolerance in Staphylococcus aureus: Influence of the alternative factor σ

The role of σ^B in the Staphylococcus aureus heat-shock induced thermotolerance was investigated. Survival curves at 58 °C of S. aureus strain Newman and its isogenic ΔsigB mutant were obtained for native and heat-shocked cells (45 °C for 5–120 min) in exponential and stationary phase of growth. The magnitude of the acquisition of thermotolerance at 58 °C depended on the growth phase and on the duration of the heat shock. Stationary growth phase cells were always more heat tolerant than exponentially growing cells and thermotolerance increased with heat-shock duration up to 120 min. S. aureus cells were able to increase their heat tolerance in the absence of the σ^B factor. In stationary phase, whereas in the parental strain the thermotolerance was increased by a factor of 12 after a heat shock of 120 min at 45 °C (δ values at 58 °C for native and heat-shocked cells were 0.63 and 7.22 min, respectively), in the mutant strain it increased 43 fold (δ values 0.09 and 3.87 min). The addition of chloramphenicol to the adaptation medium resulted in a lower increase in heat tolerance but did not prevent it completely, suggesting that S. aureus can partially increase its thermotolerance without “de novo” protein synthesis. Both the number of non-damaged cells and the proportion of cells able to repair sublethal damage were higher for heat-shocked cells.     

Comparison of antimicrobial resistance in Salmonella and Campylobacter isolated from turkeys in the Midwest USA

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest^®. In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase (int1), class 1 integrons (Salmonella and Campylobacter) and a multidrug efflux pump (Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene (int1), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1, but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.     

Genomic and phenotypic characterization of Escherichia coli isolates recovered from the uterus of puerperal dairy cows

The role of Escherichia coli in the pathogenesis of the puerperal uterine infection of the cow is largely unknown. It is proposed that E. coli favors the persistence of Arcanobacterium pyogenes and gram-negative bacteria that are pivotal to the establishment of the infection. Here, we report the genomic and phenotypic characteristics of 72 E. coli isolates recovered from the uterus of dairy cows with normal puerperium (n = 12; 35 isolates) or clinical metritis (n = 18; 37 isolates), in an attempt to identify characteristics that are related to the establishment of uterine infection. We evaluated DNA fingerprints generated by repetitive element sequence-based PCR, phylogenetic grouping, the presence of 15 virulence factor genes, in vitro biofilm formation and its relationship to curli fimbriae expression, and cellulose production. We found a wide genetic diversity (40 clonal types), including types common to normal puerperium and clinical metritis cows (n = 6), as well as types specific to normal puerperium (n = 14) or clinical metritis (n = 20) cows. Isolates were assigned to phylogenetic groups B1 (58%), A (31%), and D (11%). Only 4 virulence factor genes were detected (hlyE, hlyA, iuc, and eaeA). In vitro biofilm formation was significantly affected by culture medium and incubation temperature. Curli fimbriae expression and cellulose production, although related to biofilm formation, were not required for it. None of the evaluated E. coli characteristics were significantly related to the establishment of the uterine infection. In conclusion, data presented in this paper indicate that E. coli isolates recovered from the uterus of puerperal cows present a wide genetic diversity, do not belong to a known pathogenic group, and have a low potential of virulence and persistence. This corroborates the putative role of the bacterium in the pathogenesis of the puerperal uterine infection of the cow.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae

Isoamyl alcohol oxidase (IAAOD) is a novel enzyme that catalyzes the formation of isovaleraldehyde, which is the main component of mureka that gives sake an off-flavor (Yamashita et al. Biosci. Biotechnol. Biochem., 63, 1216–1222, 1999). We cloned the genomic DNA sequence encoding IAAOD from a koji mold, Aspergillus oryzae, using a PCR-amplified DNA fragment corresponding to the partial amino acid sequences of the purified protein as a probe. The cloned gene comprises 1903 bp of an open reading frame with three putative introns and encodes 567 amino acids with a presumed signal peptide consisting of 24 amino acids at the N-terminus. Moreover, nine potential N-glycosylation sites were present. Homology search on amino acid sequence showed that IAAOD has a region significantly similar to those conserved in FAD-dependent oxidoreductases. Southern hybridization analysis revealed that the cloned gene exists as a single copy in the A. oryzae RIB 40 chromosome. The cloned gene was overexpressed under the control of the amyB promoter in A. oryzae. The isovaleraldehyde-producing activity in the culture supernatant of one transformant was over 800 times as high as that of transformant with the control vector. This result demonstrates that the cloned gene encodes IAAOD. We named this novel alcohol oxidase gene “mreA”.