Optimization of agro‐residual medium for α‐amylase production from a hyper‐producing Bacillus subtilis KCC103 in submerged fermentation

BACKGROUND: Although submerged fermentation (SmF) is the conventional method in industry, use of low‐cost agro‐residues for α‐amylase production in SmF has not been well established. Here we optimized agro‐residue‐based medium and culture conditions for α‐amylase production in SmF using a hyper‐producing Bacillus subtilis KCC103. RESULTS: B. subtilis KCC103 produced α‐amylase in SmF by utilizing agro‐residues. Wheat bran (WB) and sunflower oil cake (SFOC) were selected as the best substrates using shake flasks. Medium containing WB (carbohydrate rich) and SFOC (rich in protein and free amino acids) at 1:1 (w/w) ratio produced high levels (90 IU mL⁻¹) of α‐amylase at 30–36 h in a shake flask. The α‐amylase yield was 14‐fold enhanced (1258 IU mL⁻¹) by optimizing process parameters and medium composition following response surface methodology in a bioreactor. The optimal conditions were: WB 1.27%, SFOC 1.42%, pH 7, 37 °C and 10–12 h. Both in shake flask and bioreactor α‐amylase synthesis was not repressed by the release of simple sugars into the medium. CONCLUSION: KCC103 with catabolite derepression and hyperproducing ability is useful for economic α‐amylase production using low‐cost agro‐residual substrates in conventional SmF. Since the production time (10–12 h) is much shorter than other strains this would improve productivity and further reduce the cost of α‐amylase production. Copyright © 2008 Society of Chemical Industry     

Production of α‐amylase under solid‐state fermentation utilizing coffee waste

Coffee industry substrates such as coffee pulp, coffee cherry husk, silver skin, spent coffee and mixtures of these coffee wastes (MC) were evaluated for their efficacy as sole carbon source for the synthesis of α‐amylase in solid‐state fermentation (SSF) using a fungal strain of Neurospora crassa CFR 308. For SSF with coffee pulp and with MC, α‐amylase activity of 3908 U g^?1 ds (units per gram of dry substrate) and 3870 U g^?1 ds, respectively, was observed. Parameters such as moisture (60%), pH (4.6), temperature (28 °C), particle size (1.0 mm), inoculum size (10⁷ spores g^?1 ds), and fermentation time (5 days) were optimized for enzyme synthesis, wherein 4981 and 4324 U g^?1 U g^?1 ds of α‐amylase activity was obtained in SSF with coffee pulp and MC, respectively. The enzyme production was further improved when the substrates were subjected to pre‐treatment by steaming. Accordingly, maximum α‐amylase activity of 7084 U g^?1 ds and 6342 U g^?1 ds was obtained with steam‐pretreated coffee pulp and MC, respectively, demonstrating them to be excellent sole carbon sources for synthesis of α‐amylase production. Copyright © 2009 Society of Chemical Industry     

Kinetics of the thermal inactivation of Bacillus subtilis α‐amylase and its application on the desizing of cotton fabrics

The thermal inactivation of Bacillus subtilis α‐amylase was studied in the presence and in the absence of Ca²⁺ at various temperatures. Inactivation rate constant (k), half‐life time (t_1/2), and activation energy (E_a) were determined to characterize the inactivation of the enzyme. Results obtained showed that the thermal inactivation of Bacillus subtilis α‐amylase followed a first‐order kinetics. The addition of Ca²⁺ had a good thermostabilizing effect on the enzyme. The stabilizing effect of Ca²⁺ is reflected by the increased values of the activation energy, which is about two times higher in the presence than that in the absence of 20 mM Ca²⁺, and the decreased values of the inactivation rate constants. The desizing of the cotton fabrics was performed through steaming at 100°C with Bacillus subtilis α‐amylase. The desizing efficiency seemed to be dependent on the concentration and pH value of the enzyme solution. It was found that through the steaming process with α‐amylase, the desizing ratio of the cotton fabrics could be beyond 98% and little damage happened to the fibers of the fabrics. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Optimization of medium and process parameters for a constitutive α‐amylase production from a catabolite derepressed Bacillus subtilis KCC103

BACKGROUND: In Bacillus subtilis KCC103 α‐amylase is hyper‐produced and not catabolite repressed by glucose. Various sugars, raw starches and nitrogen sources were tested for their repression effect on α‐amylase synthesis. Enhancement of α‐amylase production by supplementing micronutrients and surfactants was studied. Using optimized medium, process parameters were optimized for improved α‐amylase production. RESULTS: α‐Amylase was produced from KCC103 utilizing simple sugars indicating the absence of catabolite repression. Raw potato and yeast extract were best carbon and nitrogen sources for α‐amylase production. α‐Amylase synthesis was enhanced by micronutrients cysteine, thiamine, Mg²⁺ and SDS. Maximum α‐amylase (394 IU mL^?1) was produced in the optimized medium consisting of (in g L^?1) raw potato (30.0), yeast extract (20.0), cysteine (0.3), thiamine (0.2), SDS (0.2) and MgSO₄ (0.5 mmol L^?1) at 36–48 h under optimal conditions (pH 7.0, 37 °C, 200 rpm). The α‐amylase production was further enhanced to 537.7 IU mL^?1 with shorter time (15–18 h) in a bioreactor with optimized agitation rate of 700 rpm at 30% dissolved oxygen. CONCLUSION: Since there was no carbon catabolite repression of α‐amylase synthesis, sugar mixture from various agro‐residues hydrolysates could be utilized for α‐amylase production. The study showed the feasibility of utilization of raw potato for α‐amylase production from the KCC103, which would lead to a significant reduction in process cost. Copyright © 2008 Society of Chemical Industry     

Immobilization of malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

BACKGROUND: A malto‐oligosaccharide forming α‐amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α‐amylase immobilized in different supports were analyzed. RESULTS: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl₂ were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto‐oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto‐oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. CONCLUSION: The immobilized beads of α‐amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto‐oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro‐residues in a short time and immobilized enzyme can be recycled, the overall cost of malto‐oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry     

Optimization of α‐amylase production in solid substrate fermentation

Response surface methodology (RSM) was used to optimize the medium components of α‐amylase production using solid substrate fermentation (SSF). Hazelnut cake (HC), peptone, yeast extract (YE), and (NH₄)₂SO₄ were selected as independent variables for optimization. Central composite design (CCD) was used in design experiments and analysis results. This procedure limited the number of actual experiments performed while allowing possible interactions between the independent variables. By using CCD, 30 experiments were performed for determining the interaction of independent variables and optimization of fermentation medium. The P‐value of the coefficient of linear effect of (NH₄)₂SO₄ concentrations, which was obtained as 0.0001 has shown that this parameter has the greatest effect on the production of α‐amylase. Model F‐value (5.62) implies that the model is significant. The highest α‐amylase activity (4895 IU) was measured when the HC, peptone, YE, and (NH₄)₂SO₄ concentrations in the medium were 22.62, 5.20, 1.62, and 6.81 g L^?1, respectively.     

Optimization of growth medium for the production of α‐amylase from Bacillus amyloliquefaciens using response surface methodology

The optimization of nutrient levels for the production of α‐amylase by Bacillus amyloliquefaciens was carried out using response surface methodology (RSM) based on the 2³ factorial central composite design (CCD). This procedure limited the number of actual experiments performed while allowing for possible interactions between three components. RSM was adopted to derive a statistical model for the effect of starch, peptone and yeast extract (YE) on α‐amylase production. The P‐value of the coefficient for linear effects of starch and YE concentration was <0.0001, suggesting that this was the principal experimental variable, having the greatest effect on the production of α‐amylase. The optimal combinations of media constituents for maximum α‐amylase production were determined as 12.61 g L^?1 starch, 2.83 g L^?1 peptone and 1.25 g L^?1 YE. The optimization of the medium resulted not only in a 34% higher enzyme activity than unoptimized medium but also in a reduced amount of the required medium constituents. Copyright © 2006 Society of Chemical Industry     

Optimum Production and Characterization of Thermostable Amylolytic Enzymes from B. stearothermophilus GRE1

Optimum production of extracellular, thermostable amylolytic enzymes (α and β‐amylase) by a newly isolated bacterium, Bacillus stearothermophilus, was investigated in a batch bioreactor. Starch and lactose at 1.0% and 3.0% (w/v) respectively were found to be optimum for maximum enzyme production. Optimization of cultural conditions (pH 7.0 and temperature 45°C) resulted in high bacterial specific growth rate (0.64 h^?1), yielding 2.20 gL^?1 biomass, 11.43 UmL^?1 α‐amylase and 10.04 UmL^?1 of β‐amylase. Hydrolysis of native starches from wheat, cassava, corn and potato at 60°C using the crude enzyme showed 60‐80% saccharification with potato starch showing the least and wheat starch showing the greatest hydrolysis. The K_m and V_max values of the crude α‐amylase for starch were 4.78 mg starch/mL and 6.67 mg/mL.min respectively.     

Effect of non‐ionic surfactants on elimination of polycyclic aromatic hydrocarbons (PAHs) in soil‐slurry by Phanerochaete chrysosporium

Surfactants can enhance bioremediation of soil contaminated with polycyclic aromatic hydrocarbons (PAHs) by increasing their bioavailability. The objective of this study was to evaluate the effect of non‐ionic polyoxyethylene (POE) surfactants on the elimination of nine PAHs in contaminated soil by the white rot fungus Phanerochaete chrysosporium. PAHs in both liquid and solid phases were extracted and then assayed using GC–MS. Fungal biomass concentration and the activity of extracellular ligninolytic enzymes were used to determine the toxicity of POE surfactants. Results indicated that 0.5% (w/v) of surfactant Tween 80 was not toxic to fungal growth and had little impact on extracellular ligninolytic enzyme synthesis. About 80% of low ring PAHs (ie acenaphthene and fluorene) could be efficiently catabolized by P chrysosporium without surfactants, while catabolism of others (ie phenanthrene, fluoranthene, pyrene, chrysene, benzo(a)pyrene, dibenz(ah)anthracene and benzo(ghi)perylene) was less than 30% over a 8‐day culture period. Elimination of four‐to six‐ring PAHs in contaminated soil was enhanced in P chrysosporium cultures with 0.4% (w/v) Tween 80 by 15–33% compared with those in cultures without Tween 80, while no obvious effect was observed in the elimination of three‐ring PAHs over the culture period. Investigations on PAH concentrations in aqueous phase during the culture period showed that Tween 80 increased PAH aqueous concentrations and the PAH oxidation rate in aqueous was rapid. Enhancement of soil PAH elimination in the presence of Tween 80 was due to the increased PAH bioavailability. © 2001 Society of Chemical Industry     

The effects of a biosurfactant on oxygen transfer in a cyclone column reactor

A laboratory-scale cyclone column reactor was tested to determine how its oxygen transfer characteristics were affected by surfactants in the liquid medium. The volumetric oxygen transfer coefficient was greatly decreased by small quantities of the synthetic surfactants dodecyltrimethylammonium bromide and sodium dodecylsulfate, and the biosurfactant surfactin produced by Bacillus subtilis (ATCC 21332). Since the gas holdup fraction was generally increased due to foaming, the effectiveness of the surfactants was probably due to an increase in the interfacial film resistance. B. subtilis was grown in the cyclone column to 0.6 g dm^?3 with a significant level of surfactin produced while maintaining at least 75% oxygen saturation in the broth. Process optimization and scale-up of surfactin production will have to consider oxygen transfer as a key parameter.     

Lecithin‐enhanced biotransformation of cholesterol to androsta‐1,4‐diene‐3,17‐dione and androsta‐4‐ene‐3,17‐dione

A biotransformation process using Mycobacterium sp was studied for androsta‐1, 4‐diene‐3,17‐dione (ADD) and androsta‐4‐ene‐3,17‐dione (AD) production from cholesterol. Cholesterol has a poor solubility in water (～1.8 mg dm^?3 at 25 °C), which makes it difficult to use as the substrate for biotransformation. Lecithin is a mixture of phospholipids of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which behave like surfactants and can form planar bi‐layer structures in an aqueous medium. Therefore, a small amount of lecithin (<1 g dm^?3) can be used to form stable colloids with cholesterol at a relatively high concentration (20 g dm^?3) in water. In this work, an energy density of 1000 J cm^?3 from sonication was provided to overcome the self‐association of cholesterol and to generate a stable lecithin–cholesterol suspension that could be used for enhanced biotransformation. The lecithin–cholesterol suspension was stable and could withstand typical autoclaving conditions (121 °C, 15 psig, 20 min). In contrast to conventional surfactants, such as Tween 80, that are commonly used to help solubilize cholesterol, lecithin did not change the surface tension of the aqueous solution nor cause any significant foaming problem. Lecithin was also biocompatible and showed no adverse effect on cell growth. Compared with the medium with Tween 80 as the cholesterol‐solubilizing agent, lecithin greatly improved the biotransformation process in regard to its final product yield (～59% w/w), productivity (0.127–0.346 g dm^?3 day^?1), ADD/AD ratio (6.7–8), as well as the long‐term process stability. Cells can be reused in repeated batch fermentations for up to seven consecutive batches, but then lose their bioactivity due to aging problems, possibly caused by product inhibition and nutrient depletion. © 2002 Society of Chemical Industry     

Immobilization of α‐amylase in polyelectrolyte complexes

Herein, we report the formation of α‐amylase containing polyelectrolyte complexes (PECs). The method for the encapsulation of α‐amylase is based on interactions between two oppositely charged polyelectrolytes, poly(acrylic acid) (PAA) and polyethylenimine (PEI). We could show that electrostatic interactions ensure the incorporation of the enzyme into the formed polyelectrolyte complexes. The encapsulation has no negative effect on enzyme activity and protects against denaturation of the enzyme initiated by low pH values. The resulting PECs are 150–250 nm in size with narrow size distribution, appear in a spherical shape and are colloidally stable. The complexation of both polyelectrolytes and the immobilization of α‐amylase are investigated using fractionating techniques mainly the analytical ultracentrifugation and asymmetrical‐flow field‐flow fractionation. The formation of PECs represents a simple method for the encapsulation of α‐amylase without the use of organic solvents and requires no additional purifications steps. This one‐step approach, yielding high encapsulation efficiencies, shows the potential as a drug delivery system for sensitive hydrophilic actives in future. α‐amylase is immobilized in polyelectrolyte complexes made of polyethylenimine and poly(acrylic acid). Optimized encapsulation conditions and the resulting polyelectrolyte complexes are investigated via determination of IEP, α‐amylase activity assays, nanoDSC measurements, zeta potential values, dynamic light scattering, microscopy, and fractionating techniques. The encapsulated enzyme is protected against denaturation initiated by low pH values. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45036.     

Kinetic Resolution of α‐Bromoamides: Experimental and Theoretical Investigation of Highly Enantioselective Reactions Catalyzed by Haloalkane Dehalogenases

Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic α‐bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen α‐bromoamides, with different Cα‐ and N‐substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value >200). In all cases, the (R)‐α‐bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme:substrate ratio less 1:50 w/w) were all completed within 17–46 h and optically pure α‐bromoamides and α‐hydroxyamides were isolated with good yields (31–50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)‐substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (>157°) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.     

Tween80对稻草水解及同步糖化与发酵产乳酸的影响

在生物转化纤维原料产乳酸的过程中,酶解纤维原料产还原糖是限速步骤。为了获得较高的产物产率,需较高的酶用量,这使大规模酶解废弃纤维原料的成本很高。对吐温80在酶解稻草纤维素产糖,以及耐高温乳酸菌同步糖化发酵稻草产乳酸过程中的作用进行了考察。初步结果表明,吐温80加入可使保持同等程度的水解率所需的酶用量降低,添加0.2 g/g底物的吐温80到酶用量10 FPU/g体系,水解120 h的糖产率为292.2 mg/g,比不加表面活性剂体系的糖产率增加了11%;添加0.7 g/L的吐温80进行同步糖化与发酵72 h,能使乳酸产量提高24.2%。     

Batch production of biosurfactant with foam fractionation

Methods of producing the biosurfactant surfactin from cultures of Bacillus subtilis (BBK006) have been investigated. A reactor with integrated foam fractionation was designed and used in batch mode, and the performance compared with that of the same culture in shaken flasks. In the batch reactor, significant foaming occurred between 12.5 h and 14.5 h of culture time. During this period, the foam was routed through the foam fractionation column to a mechanical foam breaker, and a biosurfactant‐enriched foamate was collected. Concentration of surfactin in the foamate product was around 50 times greater than that in the culture medium. Using the integrated reactor, 136 mg L^?1 of surfactin was produced, significantly more than was achieved in shaken flasks (92 mg L^?1). The foam fractionation method allowed a real‐time measurement of the rate of surfactin production during growth. This showed that the maximum rate of production occurred at the interphase between log and stationary modes of growth, in contrast to previous work showing that surfactin is exclusively a secondary metabolite. The high value of surfactin yield in relation to biomass (Y_P/x = 0.262) indicated that surfactin was produced very efficiently by Bacillus subtilis (BBK006) in this integrated bioreactor. Copyright © 2006 Society of Chemical Industry     

Use of surface response methodology to optimize culture conditions for iturin A production by Bacillus subtilis in solid-state fermentation

Response surface methodology (RSM) was employed to optimize the cultivation conditions of Bacillus subtilis S3 for the enhancement of iturin A, a lipopeptide antibiotic used as biological pesticide, production in solid-state fermentation (SSF). The statistic experimental model predicted a maximum iturin production of 11.435 mg/g-wet solid material. Verification of the calculated maximum was done with experiments that were performed in the culture media representing the optimum combination found, and the iturin A production of 11.447 mg/g-wet solid material (average of three repeats) was obtained when B. subtilis S3 was cultivated at 25 °C for 5 days in solid fermentation containing high gluten flour 10 g and rice bran 50 g in addition to glucose 1.15%, KH₂PO₄ 1.27 mM, MgSO₄ 5.08 mM, peanut oil 1.01%, inoculum 19.49% and water content 44.97%. The iturin A production by B. subtilis S3 was increased significantly by 23%, from 9.26 mg/g-wet solid material to 11.447 mg/g-wet solid material when the strain was cultivated in the optimal medium developed by surface response methodology, as compared to medium conventionally developed by one-factor-at-a-time. The yield of iturin A (11.447 mg/g-wet solid material, with 45% moisture content) produced by B. subtilis S3 reported in this study is the highest reported to date for B. subtilis species in SSF. In addition, the use of rice bran as a substrate in solid-state fermentation for iturin A production by B. subtilis is unique.     

Sustainable Production of Biosurfactant from Agro-Industrial Oil Wastes by Bacillus subtilis and Its Potential Application as Antioxidant and ACE Inhibitor

The microbial conversion of agro-industrial oil wastes into biosurfactants shows promise as a biomass refinery approach. In this study, Bacillus subtilis #309 was applied to produce surfactin using rapeseed and sunflower cakes, the most common oil processing side products in Europe. Studies of the chemical composition of the substrates were performed, to determine the feasibility of oil cakes for surfactin production. Initially, screening of proteolytic and lipolytic activity was performed to establish the capability of B. subtilis #309 for substrate utilization and hence effective surfactin production. B. subtilis #309 showed both proteolytic and lipolytic activity. The process of surfactin production was carefully analyzed by measurement of the surfactin concentration, pH, surface tension (ST) and emulsification index (E₂₄). The maximal surfactin concentration in the sunflower and rapeseed cake medium reached 1.19 ± 0.03 and 1.45 ± 0.09 g/L, respectively. At the same time, a progressive decrease in the surface tension and increase in emulsification activity were observed. The results confirmed the occurrence of various surfactin homologues, while the surfactin C₁₅ was the dominant one. Finally, the analysis of surfactin biological function exhibited antioxidant activity and significant angiotensin-converting enzyme (ACE)-inhibitory activity. The half-maximal inhibitory concentration (IC₅₀) value for ACE inhibition was found to be 0.62 mg/mL for surfactin. Molecular docking of the surfactin molecule to the ACE domains confirmed its inhibitory activity against ACE. Several interactions, such as hydrophobic terms, hydrogen bonds and van der Waals interactions, were involved in the complex stabilization. To the best of our knowledge, this is the first report describing the effect of a lipopeptide biosurfactant, surfactin, produced by B. subtilis for multifunctional properties in vitro, namely the ACE-inhibitory activity and the antioxidant properties, using different assays, such as 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). Thus, the ACE-inhibitory lipopeptide biosurfactant shows promise to be used as a natural antihypertensive agent.     

Evaluation of production of lipopeptide biosurfactants and surfactin micelles by native Bacillus of Iran,for a broader application range

Surfactin is one of the most important lipopeptide biosurfactants obtained by biocatalysts of Bacillus subtilis. The aim of this study was to isolate surfactin-producing bacilli from native soils of the country (Iran), investigate their properties, convert surfactin to surfactin micelles, determine the properties of surfactin micelles and investigate the effect of starch-coated Fe⁰ and Fe³⁺ nanoparticles on the production of surfactin from B. subtilis. To do so, 20 bacilli were isolated from the native soil sample by heat shock method and genome sequenced by SrRNA16 method. The samples with strong β-hemolysis activity were screened as surfactant-producing strains. Two species of 61 and 63 (B. subtilis subspecies. Inaquosorum) were selected and examined by quantitative and qualitative screening tests such as hemolysis activity, surfactin production, droplet aggregation, emulsifying activity, and surface tension reduction in MSM medium containing Fe⁰ and Fe³⁺ nanoparticles. Surfactin was converted to surfactin micelles by sonication and confirmed by SEM. The antimicrobial and emulsifying activity and surface tension reduction of surfactin micelles were investigated. According to the results, the surface tension reduction of surfactin micelles was greater than that of surfactin. The strain 61 (99.7%) culture in 5 L bioreactors containing Fe³⁺ nanoparticles produced more surfactin than culture of the same strain without nanoparticles. This study presents an efficient method to increase the biosynthesis of microbial metabolites.     

One‐step production of 2,3‐butanediol from starch by secretory over‐expression of amylase in Klebsiella pneumoniae

BACKGROUND: 2,3‐Butanediol (2,3‐BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3‐BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3‐BD directly from starch. RESULTS: In this work, gene malS coding for α‐amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over‐expression of α‐amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one‐step production of 2,3‐BD from starch was carried out by engineered K. pneumoniae. A 2,3‐BD concentration of 3.8 g L^?1 and yield of 0.19 g 2,3‐BD g^?1 starch were obtained after 24 h fermentation. CONCLUSION: The one‐step production of 2,3‐BD from starch was achieved by secretory over‐expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. Copyright © 2008 Society of Chemical Industry     

Economic production of Bacillus subtilis SPB1 biosurfactant using local agro‐industrial wastes and its application in enhancing solubility of diesel

BACKGROUND: The present work aimed to optimize a new economic medium for lipopeptide biosurfactant production by Bacillus subtilis SPB1 for application in the environmental field as an enhancer of diesel solubility. Statistical experimental designs and response surface methodology were employed to optimize the medium components. RESULTS: A central composite design was applied to increase the production yield and predict the optimal values of the selected factors. An optimal medium, for biosurfactant production of about 4.5 g L^?1, was found to be composed of sesame peel flour (33 g L^?1) and diluted tuna fish cooking residue (40%) with an inoculum size of 0.22. Increased inoculum size (final OD₆₀₀) significantly improved the production yield. The emulsifier produced was demonstrated to be an alternative to chemically synthesized surfactants since it shows high solubilization efficiency towards diesel oil in comparison with SDS and Tween 80. CONCLUSION: Optimization studies led to a strong improvement in production yield. The emulsifier produced, owing its high solubilization capacity and its large tolerance to acidic and alkaline pH values and salinity, shows great potential for use in bioremediation processes to enhance the solubility of hydrophobic compounds. © 2012 Society of Chemical Industry