Production of α-amylase with Aspergillus oryzae on spent brewing grain by solid substrate fermentation

Ten Aspergillus oryzae strains were screened in solid substrate fermentation for α-amylase production on spent brewing grain (SBG) and on corn fiber. SBG proved to be a better substrate for enzyme production than corn fiber. A Plackett-Burman experimental design was used to optimize the medium composition for the best strain. Solid substrate fermentation on optimized medium with A. oryzae NRRL 1808 (=ATCC 12892) strain in stationary 500-mL Erlenmeyer flask culture yielded 4519 U of α-amylase/g of dry matter substrate in 3 d. The whole solid substrate fermentation material (crude enzyme, in situ enzyme) may be considered a cheap biocatalytic material for animal feed rations and for bioalcohol production from starchy materials.     

Kinetic characterization of extracellular α-amylase from a derepressed mutant ofBacillus licheniformis

Three strains ofBacillus licheniformis were isolated and screened for α-amylase production by solid-state fermentation. Of these, IS-2 gave relatively higher enzyme production (32±2.3 U/[g·min]) and was selected for improvement after treatment withN-methylN-nitroN-nitroso guanidine (NG) or nitrous acid (NA) to enhance its hydrolytic potential. Among the mutant variants, NA-14 gave higher enzyme production (98±1.6 U/[g·min]), and hence, was selected for kinetic and thermal characterization. M1 as a moistening agent (pH 7.0, optimized) supported 2.65-fold improved amylolytic activity by the derepressed mutant 72 h after inoculation. The values of product yield coefficient (Y _p/x=1833.3 U/g) and specific rate constant (q _p=25.46 U/[g·h]) with starch were severalfold improved over those from other carbon sources and the other cultures. The purified enzyme from NA-14 was most active at 40°C; however, the activity remained almost constant up to 44°C. The NA-induced random mutagenesis substantially improved the enthalpy (ΔH _D=94.5±11 kJ/mol) and entropy of activation (ΔS=−284±22 J/[mol·K]) for α-amylase activity and substrate binding for starch hydrolysis. The results of this study (117.8±5.5 U/[g·min]) revealed a concomitant improvement in the endogenous metabolism of the mutant culture for α-amylase production.     

Heterologous Expression of a Hyperthermophilic α-Amylase in Xanthan Gum Producing Xanthomonas campestris Cells

A hyperthermophilic α-amylase encoding gene from Pyrococcus woesei was transferred and expressed in Xanthomonas campestris ATCC 13951. The heterologous α-amylase activity was detected in the intracellular fraction of X. campestris and presented similar thermostability and catalytic properties with the native P. woesei enzyme. The recombinant α-amylase was found to be stable at 90 °C for 4 h and within the same period it retained more than 50% of its initial activity at 110 °C. Furthermore, X. campestris transformants produced similar levels of recombinant α-amylase activity regardless of the carbon source present in the growth medium, whereas the native X. campestris α-amylase production was highly dependent on starch availability and it was suppressed in the presence of glucose or other reducing sugars. On the other hand, xanthan gum yield, which appeared to be similar for both wild type and recombinant X. campestris strains, was enhanced at higher starch or glucose concentrations. Evidence presented in this study supports that X. campestris is a promising cell factory for the co-production of recombinant hyperthermophilic α-amylase and xanthan gum.     

α-amylase production by free and immobilized Bacillus subtilis

The effect of glucose on the α-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific α-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the α-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific α-amylase production with the free-cell system was produced by the immobilized-cell batch culture.     

Isolation and characteristics of highly active α-amylase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>-150

Partially purified enzyme preparation with specific activities of 153.7 U/mg for α-amylase and 0.15 U/mg for protease was produced by selective adsorption on starch. Enzymes were purified until homogeneous electrophoretically by gel-filtration over HW-55 TSK-gel with specific activities of 245 U/mg for α-amylase and 1.44 U/mg for protease. The optimum temperature and pH for purified α-amylase activity are 40–50°C and pH 6.0. The effects of various metal ions on the activity and stability of the enzyme were studied. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 374–376, July–August, 2007.     

Factors influencing activity of enzymes and their kinetics

The conventional chemically based method of dehairing and fiber-opening discharges an enormous amount of pollutants in the processing of skins. Hence, bioprocessing of skin through a two-step process, dehairing using protease and fiber opening using α-amylase, has been developed. However, because this process involves two steps, we characterized commercial protease and α-amylase for their optimum activity and determine the influence of one enzyme on the activity of the other, in order to develop an integrated enzymatic dehairing and fiber-opening process. The influence of various factors, substrate concentration, time, pH, and temperature, on the activity of both protease and α-amylase was determined. Furthermore, the activity of protease on mixing with α-amylase and vice versa was investigated. It was found that there was no significant change in the activity of one enzyme in the presence of the other. Lineweaver-Burk plots showed K _m and V _max values of 31.6 mg/mL and 0.0106 mg/(mL@min) for protease and 8.79 mg/mL and 0.0912 mg/(mL@min) for α-amylase. This study provides substantial evidence for integrating the enzyme-based dehairing and fiber-opening processes using both the selected protease and α-amylase in one step.     

Enhanced alkaline protease production in addition to α-amylase via constructing a Bacillus subtilis strain

Bacillus subtilis Bios 11 strain was previously isolated and identified. This strain naturally produces a high level of α-amylase. The multicopy (pS1) plasmid that carries the complete alkaline protease aprA gene was introduced to this host strain by transformation. The newly constructed strain was found to express the aprA gene and produces a high level of alkaline protease. The level of α-amylase production was not affected compared with the parent strain. The pS1 plasmid in the new host was proved to be segregationally and structurally stable, and the multicopy aprA gene was expressed at the stationary phase. This expression did not affect growth rate and sporulation frequency. Moreover, the level of α-amylase was maintained. Both alkaline protease and α-amylase enzymes were purified using a single-step affinity chromatography column. The use of the newly constructed strain would be valuable to the enzyme industry and would promote recycling of some food-processing wastes.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

Influence of oxygen availability on cell growth and xylitol production by Candida guilliermondii

Oxygen availability is the most important environmental parameter in the production of xylitol by yeasts, directly affecting yields and volumetric productivity. This work evaluated the cell behavior in fermentations carried out with different dissolved oxygen concentrations (0.5–30.0% of saturation), as well as a limited oxygen restriction (0% of saturation), at several oxygen volumetric transfer coefficients (12 ≤ k _L a ≤ 70 h⁻¹). These experiments allowed us to establish the specific oxygen uptake rate limits to ensure high yields and volumetric productivity. When oxygen availability was limited, the specific oxygen uptake rate values were between 12 and 26 mg of O₂/of g cell·h, resulting in a yield of 0.71 g of xylitol/xylose consumed, and 0.85 g/[L·h] for the volumetric productivity. According to the results, the effective control of the specific oxygen uptake rate makes it possible to establish complete control over this fermentative process, for both cell growth and xylitol production.     

Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production

Nutritional requirements of a strain ofBacillus thuringiensis (Bt) subsp.kurstaki were elucidated for δ-endotoxin production. The effect of some principal nutrients was deeply investigated, showing several nutritional and metabolite limitations in Bt growth and δ-endotoxin synthesis. This led us to formulate a new medium based on the hydrolysate of gruel, a cheap and abundant byproduct of semolina factories, supporting growth and δ-endotoxin synthesis. After hydrolysis of gruel by α-amylase, followed by proteolysis using alcalase, the resultant soluble material substituted glucose very well for Bt δ-endotoxin production. Indeed, 15 g/L total sugars coming from that hydrolysate, supplemented by 5.4 g/L ammonium sulfate as nitrogen source and either 5 g/L yeast extract or 3 g/L peptone from casein or 3 g/L casaminoacids or 0.25 g/L cysteine or aspartic acid, were the principal components of this new medium in which almost 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass was produced.     

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h^-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.     

Hyperthermostable,Ca<Superscript>2+</Superscript>-Independent,and High Maltose-Forming α-Amylase Production by an Extreme Thermophile <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis>: Whole Cell Immobilization

The synthesis of extracellular α-amylase in Geobacillus thermoleovorans was constitutive. The enzyme was secreted in metabolizable carbon sources as well as non-metabolizable synthetic analogues of glucose, but the titers were higher in the former than that in the latter. G. thermoleovorans is a fast-growing facultatively anaerobic bacterium that grows under both aerobic and anaerobic conditions and produces an extracellular amylolytic enzyme α-amylase with the by-product of lactic acid. G. thermoleovorans is a rich source of various novel thermostable biocatalysts for different industrial applications. α-Amylase synthesis was subject to catabolite repression in the presence of high concentrations of glucose. The addition of cAMP to the medium containing glucose did not result in the repression of α-amylase synthesis. The addition of maltose (1%) to the starch arginine medium resulted in a twofold enhancement in enzyme titers. Polyurethane foam (PUF)-immobilized cells secreted α-amylase, which was higher than that with the free cells. PUF appeared to be a better matrix for immobilization of the thermophilic bacterium than the other commonly used matrices. The repeated use of PUF-immobilized cells was possible over 15 cycles with a sustained α-amylase secretion. The use of this enzyme in starch saccharification eliminates the addition of Ca²⁺ in starch liquefaction and its subsequent removal by ion exchangers from the product streams.     

Lactobacillus plantarum amylase acting on crude starch granules

The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and nongelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.     

Effect of cotton-seed biocidal peptides and gossypol on resistance to biotic factors

Peptide fractions were isolated from seeds of eight cotton varieties differing in resistance to fungal pathogens and cotton bollworm. Their ability to inhibit the growth of V. dahliae fungal conidiae and α-amylase from various sources was studied. A correlation was found between the fungicidal activity of the peptides and the resistance of the cotton varieties to the pathogens. α-Amylase of cotton bollworm was inhibited less by the isolated peptides than α-amylase of other insects that are not cotton pests. It was shown for the first time that gossypol inhibits α-amylase of insects. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 186-188, March-April, 2009.     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon material balances

Xylitol production by Debaryomyces hansenii NRRL Y-7426 was performed on synthetic medium varying the initial xylose concentration between 50 and 300 g/L. The experimental results of these tests were used to investigate the effect of substrate level on xylose consumption by this yeast. Satisfactory values of product yield on substrate (0.74–0.83 g/g) as well as volumetric productivity (0.481–0.694 g/L·h) were obtained over a wide range of xylose levels (90–200 g/L), while a worsening of kinetic parameters took place at higher concentration, likely due to a substrate inhibition phenomenon. The metabolic behavior of D. hansenii was studied, under these conditions, through a carbon material balance to estimate the fractions of xylose consumed by the cell for different activities (xylitol production, biomass growth, and respiration) during the lag, exponential, and stationary phases.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Characteristics and production of thermostable α-amylase

Thermostable α-amylases have application in a variety of industrial processes and enzymes from a substantial number of thermophilic bacteria and fungi have been screened and characterized to varying degrees. The characteristics of these enzymes are summarized in this review. The genetics of α-amylase production inBacillus subtilis is reviewed and classical and recombinant DNA approaches to increasing α-amylase production are discussed.     

Oxygen uptake rate in production of xylitol by Candida guilliermondii with different aeration rates and initial xylose concentrations

The global oxygen uptake rate (OUR) and specific oxygen uptake rates (SOUR) were determined for different values of the volumetric oxygen mass transfer coefficient (15, 43, and 108 h⁻¹), and for varying initial xylose concentrations (50, 100, 150, and 200 g/L) in shaking flasks. The initial cell concentration was 4.0 g/L, and there was only significant growth in the fermentation with the highest oxygen availability. In this condition, OUR increased proportionally to cell growth, reaching maximum values from 2.1 to 2.5 g of O₂/(L·h) in the stationary phase when the initial substrate concentration was raised from 50 to 200 g/L, respectively. SOUR showed different behavior, growing to a maximum value coinciding with the beginning of the exponential growth phase, after which point it decreased. The maximum SOUR values varied from 265 to 370 mg of O₂/(g of cell·h), indicating the interdependence of this parameter and the substrate concentration. Although the volumetric productivity dropped slightly from 1.55 to 1.18 g of xylitol/(L·h), the strain producing capacity (γ _P/X ) rose from 9 to 20.6 g/g when the initial substrate concentration was increased from 50 to 200 g/L. As for the xylitol yield over xylose consumed (γ _P/S ), there was no significant variation, resulting in a mean value of 0.76 g/g. The results are of interest in establishing a strategy for controlling the dynamic oxygen supply to maximize volumetric productivity.     

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.