Increased production of β-cyclodextrin using an aqueous two-phase system

Summary -Cyclodextrin(-CD) was produced by cyclodextrin glycosyltransferase(CGTase) in aqueous two-phase system. -CD production from soluble starch was catalyzed by CGTase in dextran-rich bottom phase, and the -CD produced was transferred to PEG-rich top phase in aqueous two-phase system, composed of 7% (w/w) polyethylene glycol(Mr 20,000) and 10% (w/w) dextran(Mr 38,900). Partition coefficients of -CD and CGTase were 1.5 and 0.25, respectively. The total productivity of -CD in aqueous two-phase system was about 3 times of that in dextran phase.     

α-amylase production in aqueous two-phase systems with Bacillus subtilis

The production of α-amylase (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) by Bacillus subtilis has been studied in repeated batch fermentations in aqueous two-phase systems. In a phase system composed of PEG 600, 8% (w/w), PEG 3350, 5% (w/w)/Dextran T 500, 2% (w/w), 82% of the enzyme partitioned to the top phase. The enzyme concentration in the top phase reached 0.85–1.35 U ml^?1 during the fermentations compared with 0.58 U ml^?1 in the reference fermentation. In the phase system composed of PEG 3350, 9% (w/w)/Dextran T 500, 2% (w/w), 73% of the enzyme partitioned to the top phase. However, the enzyme concentration in this phase system reached only 0.35 U ml^?1 in the top phase. The bacterial cells were microscopically observed to partition totally to the bottom phase in the aqueous two-phase system used. The results are discussed in relation to recirculation of cells by immobilizing to a solid matrix. Extraction of the product to the top phase and the effect of the phase polymers, especially PEG, on the production are also discussed.     

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase. AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.     

Solid state fermentation for production of α-amylase byBacillus megaterium 16M

Summary The ratio of buffer to wheat bran, incubation temperature and initial pH influence -amylase production byBacillus megaterium 16M under solid state fermentation. The enzyme, with pH and temperature optima at 6.0 and 70°C, is formed at a level of 30,000 units/g dry bacterial bran without coproduction of proteases and cellulases.     

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.     

Rapid process for purification of an extracellular β-xylosidase by aqueous two-phase extraction

A rapid process for purification of an extracellular β-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na₂HPO₄. The aqueous two-phase extraction was performed with PE62 (10%)–NaH₂PO₄ (15%) as phase-forming reagent. SDS–PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.     

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.     

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.     

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.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha

To check feasibility and effectiveness of the α-amylase reporter system, two vectors were designed and tested using hepatitis B virus surface antigen (HBsAg) and Homo sapiens granulocyte-macrophage colony stimulating factor 2 (hGM-CSF2) as a model. By integrating the vector containing two independent cassettes into the same genome locus, high-producing clones of HBsAg (or hGM-CSF2) were screened using the α-amylase as a reporter. Results show there was a positive correlation (Correlation coefficient, R ² > 0.95) between the yield of recombinant proteins and the α-amylase activity of corresponding transformants, which was independent of the gene dosage.     

Parametric optimization of extracellular α-amylase production by thermophilicBacillus coagulans

The culture parameters required for optimum production of extracellular α-amylase by the thermophilicBacillus coagulans are described. The optimum pH, temperature and incubation period for amylase production were 7, 50°C and 48 h, respectively. Age of inoculum (48 h) and its level, (2%) were critical for maximum amylase yield. The enzyme secretion was high in rice starch and beef extract as compared to other carbon and nitrogen sources tested. The addition of mustard oil cake (1%) and agitation at 1.7 Hz resulted in an enhancement of α-amylase secretion.     

Direct screening for high-level expression of an introduced α-amylase gene in plants

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature -amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for -amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.     

Truncated α-amylase: an improved candidate for textile processing

Abstract

Enzymes are indispensable biocatalysts required in various steps of textile processing to minimize various chemical-induced hazards. The present work focuses on the applications of the truncated α-amylase in textile industry for desizing of fabrics by starch hydrolysis. The multiple sequence alignment was performed to find homology and the possible truncation region in Bacillus subtilis MTCC 121 α-amylase with same bacilli family α-amylase. Two constructs were generated for α-amylase gene of Bacillus subtilis MTCC 121 (Amy_F, full-length and Amy_T, C-terminal truncated) were cloned, overexpressed, purified, and characterized. Results revealed that activity of Amy_T was found to be 2.87-fold better than Amy_F. Further, the optimum temperature of Amy_F and Amy_T was obtained at 45?°C and 55?°C, respectively, whereas optimum pH was recorded at pH 7 and pH 8, respectively. Improved thermostability of Amy_T was further confirmed through thermal shift assay. Subsequently, starch-coated fabrics were tested for starch removal using the α-amylases. Comparative analysis revealed that Amy_T performed better in starch removal from polystyrene (85%), silk (75%), and cotton (70%) fabrics. The removal of starch from the fabrics was further confirmed by FESEM. Conclusively, this work presents one truncated α-amylase as an improved candidate over its full-length counterpart for textile desizing.     

A cell-free system for the study of α-amylase synthesis in barley aleurone layers

1. A cell-free system capable of alpha-amylase synthesis has been obtained from the aleurone layers of germinating barley. 2. This system requires potassium chloride, sucrose and an amino acid mixture in order to function. The crude preparation does not require calcium chloride. Chloramphenicol inhibits alpha-amylase synthesis as indicated both by increase in measurable enzyme activity and incorporation of l-[U-(14)C]glutamic acid.     

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.     

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