Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Control of ��-amylase production by Bacillus subtilis

This study proposes two adaptive control algorithms for the fed-batch production of α-amylase. The first one uses online information from hardware measuring glucose. Online information of both biomass and glucose concentrations measured with different frequency is used in the second algorithm. Hardware measuring variables are inputs for software sensors of glucose concentration and (specific) glucose consumption rate. Either of the algorithms do not require any kinetic coefficients. This is a benefit, because the kinetic coefficients can vary during cultivation and between cultivations, leading to low process reproducibility and the non-stationary state of the bioprocess. The results of simulation investigations show good performance of the proposed control schemes.     

Enhancement of α-amylase production by immobilized Bacillus subtilis in an airlift fermenter

Bacillus subtillis ATCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consisting of bubbling air into a round flask, the other involving sparging of air into an airlift fermenter. The latter system, which produces microbubbles, gave 40–70% increase in enzyme production over the former and a doubling of bacterial density within the beads was measured. The use of CaCl₂instead of KCl as polymerization agent led to a better yield of α-amylase.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Industrial production of α-amylase by genetically engineered Bacillus

A genetically engineered Bacillus subtilis strain (ALKO 84) has been introduced for industrial production of α-amylase. This strain carries the α-amylase gene from a traditionally developed production strain B. amyloliquefaciens (ALKO 89) on the multicopy plasmid pUB110.⁸At laboratory scale the recombinant strain ALKO 84 produced in industrial medium about twice as much α-amylase as the traditional strain ALKO 89. The process for production of the enzyme was scaled-up to 60m³. At this scale B. subtilis ALKO 84 retained its relative superiority to B. amyloliquefaciens ALKO 89, producing about 85% of the activity obtained at laboratory scale. Stability of the recombinant plasmid was found acceptable during the large-scale cultivations with over 90% of cells retaining plasmid-encoded characteristics throughout.     

Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens

Summary Bacillus amyloliquefaciens 321S cells were immobilized with 3.4% -carrageenan gel in bead form, and -amylase production by the immobilized cells was studied. Cells in the gel, after the population reached maximum were restricted to a layer of 50 m thickness, from the surface of the gel, suggesting that oxygen diffusion is the growth limiting factor. The specific respiratory activity and the growth rate of the entrapped cells under such conditions were 1/2 and 1/5 1/10, respectively, that of free cells. In spite of the repressed respiration and growth, the specific rate of -amylase production of the entrapped cells reached the maximum value of free cells or higher.In continuous culture, in an aerated vessel with a volume ratio of gel beads to medium of 1:2, the maximum production rate of -amylase was obtained at a dilution rate of 1.0 h^–1, which was double the maximum specific growth rate of the strain.These results showed that bacterial -amylase production, which is a nongrowth-associated type of synthesis was achieved with the use of immobilized cells.     

Optimization of β-mannanase production by Bacillus subtilis US191 using economical agricultural substrates

The Bacillus subtilis US191 strain producing highly thermostable β-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular β-mannanase production by this strain was 1.48 U ml⁻¹. To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett–Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on β-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box–Behnken design. Under optimized conditions (1 g L⁻¹ LBG, 8 g L⁻¹ soymeal, temperature of 30°C and inoculum size of 10¹⁰ CFU ml⁻¹), a 2.59-fold enhancement in β-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L⁻¹) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a β-mannanase production level of 8.75 U ml⁻¹ in presence of 0.25 g L⁻¹ LBG, 5 g L⁻¹ of coffee residue powder, 5 g L⁻¹ of date seeds powder, and 5 g L⁻¹ of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in β-mannanase production by B. subtilis US191 was achieved.     

Efficient production of human α-amylase by a Bacillus brevis mutant

Summary A cDNA for mature human salivary -amylase was directly joined to a sequence encoding the signal peptide of the middle wall protein (MWP) gene of Bacillus brevis 47. This hybrid gene was placed downstream from the multiple promoter region of the MWP gene on a low copy-number plasmid vector, pHW1. B. brevis 47 carrying the plasmid produced 0.9 mg/l of active human -amylase in the medium. A B. brevis 47 mutant obtained on mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine produced an increased amount of the -amylase (6 mg/l). When the fused gene was inserted into a high copy-number expression vector, pNU200, and then introduced into the mutant, a large amount (60 mg/l) of the -amylase was produced in the medium. The -amylase showed approximately the same specific activity and molecular weight as those of the natural enzyme. The mutant showed higher sensitivity to various antibiotics than the original strain, and altered cell wall and cytoplasmic membrane protein compositions. The results of reversion analysis suggested that a single mutation is responsible for the above phenotypes and hyper-productivity of human -amylase. Offprint requests to: H. Yamagata     

Influence of succinic acid on the extracellular α-amylase production by chemostat-crow Bacillus licheniformis

Summary An 8-fold increase in -amylase production by pulsing of succinic acid to a chemostat culture ofBacillus licheniformis has been shown. The -amylase concentration was found to be at the highest value two doubling times after the addition, indicating that the effect may be due to regulatory control.     

Optimization of α-amylase and glucoamylase production by recombinant strains ofSaccharomyces cerevisiae

Summary Replacement of the regulatory sequence of theBacillus amyloliquefaciens α-amylase gene (AMY1) by the yeast alcohol dehydrogenase gene promoter (ADC1 _p) resulted in increased levels of extracellular α-amylase production inSaccharomyces cerevisiae. Negative regulation of glucoamylase synthesis by theSTA10-encoded repressor was alleviated by replacing the nativeSTA2 gene promoter fromS. cerevisiae var.diastaticus withADC1 _p. Enhanced degradation of starch was achieved when the modified versions of theAMY1 andSTA2 genes were introduced jointly intoS. cerevisiae.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

OPTIMIZATION OF α-AMYLASE PRODUCTION BY Bacillus subtilis RSKK96: USING THE TAGUCHI EXPERIMENTAL DESIGN APPROACH

In this study, the Taguchi experimental design was applied to optimize the conditions for α-amylase production by Bacillus subtilis RSKK96, which was purchased from Refik Saydam Hifzissihha Industry (RSHM). Four factors, namely, carbon source, nitrogen source, amino acid, and fermentation time, each at four levels, were selected, and an orthogonal array layout of L₁₆ (4⁵) was performed. The model equation obtained was validated experimentally at maximum casein (1%), corn meal (1%), and glutamic acid (0.01%) concentrations with incubation time to 72 h in the presence of 1% inoculum density. Point prediction of the design showed that maximum α-amylase production of 503.26 U/mg was achieved under optimal experimental conditions.     

Integration,amplification and expression of the Bacillus licheniformis α-amylase gene in Bacillus subtilis chromosome

We have introduced the α-amylase gene from Bacillus licheniformis (amy gene) in a non-replicative plasmid which can be conveniently integrated and amplified at a specific site of the B. subtilis chromosome. Although we were able to select spontaneous and stable gene amplification of about 20 integrated copies, the amylase secretion remained very low. A DNA fragment presenting a high promoter activity in B. subtilis was therefore inserted upstream from the amy gene coding sequence, leading to a significant increase of amylase production. However, the amplified structures obtained with this construction were found to contain no more than 12 copies of the amy gene and to be rather unstable when cells were grown under non-selective conditions.     

Acidification of the Culture Medium by Products of Glucose Metabolism Inhibits the Synthesis of Heterologous Extracellular α-amylase by Bacillus subtilis 168

Applied Biochemistry and Microbiology - The synthesis of α-amylase AmyM3 from B. flexus 406 in the recombinant strain B. subtilis 168-28 was significantly repressed during submerged...     

The production of α-amylase in batch and chemostat culture by Bacillus stearothermophilus

The production of -amylase (-1,4-glucan 4-glucanohydrolase; EC. 3.2.1.1) by a strain of Bacillus stearothermophilus isolated from leaf litter was investigated in a tryptone-maltose medium at 55°C in batch and chemostat culture. Amylase production was growth-limited and restricted to the exponential phase in batch culture. The enzyme yield was reduced by 40% when the culture pH was maintained at pH 7.2. Amylase production in chemostat culture was influenced by the growth rate throughout the dilution rate range used.     

Hybrid on-line optimal control strategy for producing α-amylase by Bacillus subtilis

The combined effect of macronutrients in the extraction medium on α-amylase produced by Bacillus subtilis were studied by using response surface methodology in shaken flask cultures. The production of amylase was significantly affected by the interaction between wheat bran and the cotton seed extract in the extraction medium and by the interaction between the cotton seed extract and starch. The optimal combination in the extraction medium for maximum α-amylase production was determined as 10.80 g·L?1 of wheat bran, 9.90 g·L?1 of cotton seed extract, 0.5 g·L?1 of starch, 2.0 g·L?1 of yeast extract, 5.00 g·L?1 of NaCl and 2.00 g·L?1 of CaCl?. A 12.55-fold increase of enzyme activity was recorded in the optimized medium compared to the result acquired in a minimum essential medium. The optimized medium was used to compare different cultivation strategies in fermenters. The pH-stat strategy for reducing cellular stress response and the substrate concentration-stat strategy for reducing substrate inhibition were independently investigated. The temperature-limited strategy has been proposed to solve the proteolytic digestion problem, although the high-pressure strategy resulted in high productivity. A hybrid strategy simultaneously controlling pH, temperature, substrate concentration and pO? was finally investigated to enhance the efficiency of the process. This hybrid strategy resulted in high activity of α-amylase, increasing the productivity almost three-fold as compared to an ordinary fed-batch culture.     

Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Parametric analysis of metabolic fluxes of α-amylase and protease-producing Bacillus subtilis

Parametric analysis was applied for a metabolic flux model for the fed-batch culture of Bacillus subtilis producing recombinant α-amylase and protease. The metabolic flux model was formulated as a linear programming problem consisting of 49 reactions (decision variables) and 50 metabolites (equality constraints). This study was aimed to determine the response of the metabolic fluxes and objective function value of minimizing the difference between ATP consumption and ATP production (ATP balance). With regard to intracellular metabolite accumulation, the objective function value was least sensitive to variation in succinate and most sensitive to variation in malate. Amongst the variations in the accumulation rates of extracellular metabolites, the objective function value was least sensitive to variation in glutamate and most sensitive to variation in starch hydrolysis and triglyceride synthesis. A 10% variation in metabolite accumulation rates caused a maximum of 13.8% variation (standard error = 3.8%) in the objective function value.     

Stability during fermentation of a recombinant α-amylase plasmid in Bacillus subtilis

Summary We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus -amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec ⁺ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable.