Efficient extracellular expression of <Emphasis Type="Italic">Bacillus deramificans</Emphasis> pullulanase in <Emphasis Type="Italic">Brevibacillus choshinensis</Emphasis>

In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 10³ U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.     

Isolation of Aureobasidium pullulans and the effect of different conditions for pullulanase and pullulan production

Isolation and production of pullulahase by a new Aureobasidium pullulans isolate from the Fayoum Governorate (AUMC 2997) which was identified by the Assiut University Mycological Center was investigated. Another isolate from the Aswan Governorate (AUMC 1695) was kindly provided by the Assiut University Mycological Center. Acetone 2× gave better results for the precipitation of protein than 80% ammonium sulfate in the case of the media containing yeast extract. Very low protein production occurred in media without yeast extract. No enzyme production occurred in the first two days and the production of the enzyme started on the third day. Statistical analysis determined that the optimum conditions for the production of pullulanase were: incubation at 25°C for 5 days, pH 5.5, with sucrose as carbon source at 100 g/L and sodium nitrate as nitrogen source at 2 g/L. Addition of manganese chloride to the medium (1, 2 and 3 g/L) caused inhibition of pullulanase. Also, while the lowest pullulan + pigment concentrations were attained at the fifth day, pH 5.5, at 15°C, 100 g/L sucrose, 2 g/L nitrogen sources, the pullulan + pigment production increased with increasing the concentrations of manganese chloride.     

Regulation of extracellular protease production in Candida lipolytica

Production of extracellular protease by Candida lipolytica NRRL Y-1094 was depressed upon transfer to carbon-, nitrogen- or sulphur-free medium but not upon transfer to phosphorus-free medium. The protease activities produced under the three nutrient limitations had alkaline pH optima and similar substrate and inhibitor specificities. Any one of the following three conditions wass found to be sufficient for depression of extracellular protease: (1) “poor” carbon source, (b) cysteine intracellular pool below 0.5 μmol/g dry weight cells and (c) ammonia intracellular pool below 10 μmol/g dry weight cells. Thus, extracellular protease production in C. lipolyutica was subject to at least three different regulatory controls, carbon, sulphur and nitrogen repression. Intracellular cysteine and ammonia appeared to be the metabolic signals for sulphur and nitrogen repression, respectively. Anabolic glutamate dehydrogenase did not act as a regulatory protein mediating nitrogen repression. Exogenous protein had an inductive effect on extracellular protease production.     

Mutations affecting extracellular protease production in the filamentous fungusAspergillus nidulans

The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.     

Enhancement of extracellular pullulanase production from recombinant Escherichia coli by combined strategy involving auto-induction and temperature control

Pullulanase was extracellularly produced with an engineered Escherichia coli with a combined strategy. When auto-induction instead of isopropyl β-d-1-thiogalactopyranoside (IPTG) induction method was implemented, we observed increased extracellular activity (4.2 U ml^?1) and cell biomass (7.95 g DCW l^?1). Subsequent investigation of temperature effect on fermentation showed cultivation performed at 25 °C presented the highest extracellular titer and cell biomass. In order to reduce the extended production period, we developed a two-stage temperature control strategy. Its application not only reduced the production period from 72 to 36 h, but also further enhanced the yield of extracellular pullulanase. Finally, with a view to releasing more intracellular pullulanase, we altered cell membrane permeability with various medium additives. As a result, extracellular titer was elevated to 68.23 U ml^?1, nearly 35-fold higher than that with IPTG induction method. The combined strategy developed here may be useful for the production of other extracellular proteins by recombinant E. coli.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Carbon source impact on Yarrowia lipolytica KKP 379 lipase production

The production of lipases by microorganisms is strongly influenced by the culture conditions. The optimum culture conditions for enzyme production are strain- and species-dependent. The aim of this study was to evaluate the impact of the carbon source used in the culture medium on the profile of lipases produced by Yarrowia lipolytica KKP 379. We observed a different pattern of extracellular and cell-bound lipase production, which was the highest in the early exponential phase. The extracellular lipase activity increased in the late exponential phase due to the lower accumulation of lipase molecules in cell walls. The best carbon source for extracellular lipase production by Y. lipolytica KKP 379 was olive oil. Glucose, dodecane and olive oil had a positive effect on biomass yield. Dodecane and/or glycerol utilization in microbiological lipase production was possible, but this process could not proceed without the addition of some activators such as olive oil in the cultivation medium.     

Dimorphism inYarrowia lipolytica: Filament formation is suppressed by nitrogen starvation and inhibition of respiration

In contrast toSaccharomyces cerevisiae, nitrogen starvation inhibited formation of hyphae in liquid cultures ofY. lipolytica, while carbon source did not seem to be important for filament formation. Inhibitors of mitochondrial respiration strongly suppressed the development of hyphae, indicating that energy conversion processes, and thus carbon metabolism, may be involved. pH of the medium also strongly affected the morphology, but only in the presence of a complex nitrogen source, implying that the cells respond to altered nutrition in media with different pH rather than to pH itself. The results suggest that theXPR2 gene encodingY. lipolytica alkaline extracellular proteinase is involved in the regulation of dimorphism in this species.     

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

Summary A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K _m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca²⁺. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups. Offprint requests to: B. C. Saha     

A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5′ end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61–65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using β-lactamase as a reporter, we show that the FLD1 promoter (P_FLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of β-lactamase produced under control of P_FLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (P_AOX1). Thus, P_FLD1 is an attractive alternative to P_AOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.     

Effect of nutrition of Monascus sp. on formation of red pigments

Summary A higher producer of ascospores and pigments, Monascus strain TTWMB 6042, was used to study regulation of pigment production by nutrients. An initial medium containing 4% glucose, 0.3% NH₄NO₃ (75 mm nitrogen) and inorganic salts was used. We found that the formation of red pigments in this strain, measured by optical density at 500 nm (OD₅₀₀) was strongly stimulated by monosodium glutamate (MSG) as the sole nitrogen source. The choice of carbon source and an initial pH of pH 5.5 were also important. High concentrations of phosphate and MgSO₄ were inhibitory to pigment production. A new chemically defined medium was devised containing 5% maltose, 75 mm MSG, phosphate and MgSO₄ at lower concentrations plus other mineral salts, which yielded a tenfold increase in OD₅₀₀ and a reversal of the pigment location from predominantly cell-bound, including both intracellular and surface-bound pigments, to mainly extracellular. Offsprint requests to: A. L. Demain     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima,protease and ion sensitivities

Summary The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono--lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to -endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.     

Fermentative production of spiramycins

Fermentative production of spiramycins by Streptomyces ambofaciens has been performed using fermentation media of different chemical compositions. Medium I was selected from nine media as the best for production of high titres of spiramycins. Biochemical changes which occurred during fermentative production of spiramycins revealed that adjustment of the initial pH value of the medium was very important. The initial pH value of the fermentation medium which allowed the organism to produce a good yield of antibiotic was 6.5. The fermentation period affected the formation of spiramycins, and the maximum incubation period required for the fermentation process was 120 h. The role of inoculum on spiramycin yield showed that it was better to inoculate the fermentation medium with vegetative cells of Streptomyces ambofaciens rather with spores. The carbon source influenced spiramycin biosynthesis: dextrin was the best carbon source and stimulated the organism to form high titres of antibiotics. The best concentrations of dextrin and glucose for increased antibiotic yields were 25 and 15 gl^?1, respectively. Organic sources in the fermentation medium were more efficient than inorganic nitrogen sources for spiramycin formation. Fodder yeast was the best organic nitrogen source in fermentative production of spiramycins. The maximal concentrations of fodder yeast, soybean meal, peptone, Ca(NO₃)₂ and NH₄NO₃ for increased antibiotic yield were 6.5, 6.0, 4.0, 10.0 and 4.0 gl^?1, respectively.     

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase. Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.     

Mineralization of the Cyclic Nitramine Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia and Williamsia spp.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-¹⁴C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-¹⁴C]RDX to ¹⁴CO₂ was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH₄)₂SO₄ greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.     

Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production byBacillus stearothermophilus G-82

Cell growth and extracellular pullulanase production ofBacillus stearothermophilus G-82 were investigated in batch culture using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell growth in batch culture was better under oxygen unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation took place in the late growth phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of oxygen limitation.     

Impeller types and feeding modes influence the morphology and protein expression in the submerged culture of<Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

The influences of impeller types on morphology and protein expression were investigated in a submerged culture ofAspergillus oryzae. The impeller types strongly affected mycelial morphology and protein production in batch and fed-batch fermentations. Cells that were cultured by propeller agitation grew in the form of a pellet, whereas cells that were cultured by turbine agitation grew in a freely dispersed-hyphal manner and in a clumped form. Pellet-grown cells showed high levels of protein production for both the intracellular heterologous protein (β-glucuronidase) and the extracellularly homologous protein (α-amylase). The feeding mode of the carbon source also influenced the morphological distribution and protein expression in fed-batch fermentation ofA. oryzae. Pulsed-feeding mainly showed high protein expression and homogeneous distribution of pellet whereas continuous feeding resulted in less protein expression and heterogeneous distribution with pellet and dispersed-hyphae. The pellet growth with propeller agitation paralleling with the pulsed-feeding of carbon source showed a high level of protein production in the submerged fed-batch fermentation of recombinantA. oryzae.     

Purification,Crystallization and Some Properties of Intracellular Pullulanase from Aerobacter aerogenes

Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s_{20, w} was 6.29 S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1) × 10,000 from gel filtration with Sephadex G-200 and to be (9±1) × 10,000 from sedimentation equilibrium. These values were higher than that (6~7 × 10,000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.