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.     

Active site studies on Bacillus amyloliquefaciens α-amylase (I)

Summary Modification of liquefying -amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 ° C completely inactivated a-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate was one, thus indicating modification of a single histidine per mole of the enzyme. Diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH₂OH at 30 °C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the -amylase active site.     

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.     

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.     

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.     

Improved thermostable α-amylase activity of Bacillus amyloliquefaciens by low-energy ion implantation

Thermostable α-amylase is of great importance in the starch fermentation industry; it is extensively used in the manufacture of beverages, baby foods, medicines, and pharmaceuticals. Bacillus amyloliquefaciens produces thermostable α-amylase; however, production of thermostable α-amylase is limited. Ion-beam implantation is an effective method for mutation breeding in microbes. We conducted ion-beam implantation experiments using two different ions, Ar(+) and N(+), to determine the survival rate of and dose effect on a high α-amylase activity strain of B. amyloliquefaciens that had been isolated from soil samples. N(+) implantation resulted in a higher survival rate than Ar(+) implantation. The optimum implantation dose was 2.08 × 10(15) ions/cm(2). Under this implantation condition, we obtained a thermally and genetically stable mutant α-amylase strain (RL-1) with high enzyme activity for degrading α-amylase. Compared to the parental strain (RL), the RL-1 strain had a 57.1% increase in α-amylase activity. We conclude that ion implantation in B. amyloliquefaciens can produce strains with increased production of thermostable α-amylase.     

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     

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.     

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.     

Continuous production of α-amylase byBacillus amyloliquefaciens in a two-stage fermentor

Summary The production of a secondary metabolite (-amylase) by a highly aerobic bacterium (Bacillus amyloliquefaciens) was examined in batch, single-stage chemostat, two-stage stirred tank, and two-stage stirred tank/tubular reactor configurations. The relative performance of these reactor systems as measured by product concentration and volumetric productivity was compared, and the effect of aeration rate on the extent of plug flow in the tubular reactor was examined.     

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.     

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.     

Thermostable liquefying α-amylase fromBacillus amyloliquefaciens

Summary An extracellular -amylase is purified to homogeneity with 62 % recovery of the enzyme activity using heat treatment, ion-exchange and gel filtration chromatographies. The purified enzyme has a molecular weight of 68, 000, isoelectric point 6.25, optimal activity at pH 6 and temperature 65 °C, k_est 8.8×10⁸ s¹ liquefying amylase units, and K_m for starch 2.9 mg ml^–1. The enzyme is further characterized for its endo action on the starch and related polymers. Calcium stabilizes the active conformation of the enzyme during prolonged exposure to the extremes of pH and temperature. The enzyme retains 100 % activity for 24 h at 65°C and exhibits a half-life of 9, 3, and 0.5 h at 80°, 85° and 90°C, respectively.     

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.     

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.     

Banana waste as substrate for α-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation

 Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of α-amylase when banana fruit stalk was used as substrate in a solid-state fermentation system. The effects of initial moisture content, particle size, cooking time and temperature, pH, incubation temperature, additional nutrients, inoculum size and incubation period on the production of α-amylase were characterised. A maximum yield of 5 345 000 U mg^-1 min^-1 was recorded when pretreated banana fruit stalk (autoclaved at 121 °C for 60 min) was used as substrate with 70% initial moisture content, 400 μm particle size, an initial pH of 7.0, a temperature of 35 °C, and additional nutrients (ammonium sulphate/sodium nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with banana fruit stalk medium compared to wheat bran. Received: 18 April 1995/Received last revison: 6 May 1996/Accepted: 9 May 1996     

Production of α-amylase by Bacillus amyloliquefaciens in batch and continuous culture using a defined synthetic medium

Summary Using a totally defined synthetic medium the effect of lactose and nitrogen on cell physiology and -amylase production by Bacillus amyloliquefaciens B155 were investigated. Results showed cell growth and -amylase production patterns to be similar regardless of the limiting nutrient and suggested stationary phase gene control of -amylase production as opposed to a direct response to nutrient limitation.     

α-Amylase production by Bacillus amyloliquefaciens in a membrane recycle bioreactor

A strain of Bacillus amyloliquefaciens was cultivated in a membrane recycle bioreactor for the production of an extracellular -amylase. Continuous cultivations of B. amyloliquefaciens in the recycle fermentor led to higher cell mass and volumetric productivities than the ones obtained in batch or chemostat cultivations; the -amylase activities were lower than in the batch mode but significantly higher than in the chemostat mode. The operation of the membrane recycle bioreactor was sometimes disturbed by high broth viscosity leading to a stronger fouling of the membrane.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.