Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters

Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30°C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 × 10⁵ U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31°C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 × 10⁵ U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 × 10⁵ U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48–56, 1977].     

Medium optimization of a hydrophilic solvent‐stable protease from Serratia sp. SYBC H

Two statistical methods were used for medium optimization for a hydrophilic solvent‐stable protease production by Serratia sp. SYBC H with duckweed as the nitrogen source. Orthogonal design was applied to find the significant variables, then response surface methodology (RSM), including Box–Behnken central composite experiments, was used to determine the optimal concentrations and interaction of the significant variables. Results demonstrated that duckweed powder, wheat flour, Tween 80, sodium chloride had significant effects on the solvent‐stable protease production. The interaction between duckweed and wheat flour was significant. The optimal level of the variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, sodium chloride 0.08 M, Tween 80 1% v/v, initial pH 11.0, and inoculum size 7% v/v. The maximum protease activity reached 1922.8 U/mL in the optimized medium, with about 18.3‐fold higher than that in the unoptimized medium. Most importantly, the protease from Serratia sp. SYBC H has successfully catalyzed the specific acylation of sucrose in a two‐solvent medium consisting of pyridine and n‐hexane (1:1, v/v), and non‐specific acylation of sucrose in anhydrous DMSO. These results demonstrated that the protease from Serratia sp. SYBC H is a solvent‐stable protease and it could be an ideal biocatalyst for sugar esters syntheses in non‐aqueous media.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Alkaline protease production by Basidiobolus (N.C.L. 97.1.1): effect of 'darmform' morphogenesis and cultural conditions on enzyme production and preliminary enzyme characterization

A strain of Basidiobolus (N.C.L. 97.1.1) was isolated from plant detritus which secreted alkaline protease optimally active at pH 10.0. It is the first report of a protease from Basidiobolus, which is stable to and active under high alkaline conditions. When incubated under stationary conditions in broth cultures containing salts such as ammonium chloride, 'darmform' morphogenesis was readily induced through enlargement and internal division of the hyphal segments. Secretion of high activity alkaline protease was obtained in cultures initiated with darmform morphogenesis whereas cultures initiated from mycelial inocula grew as large pellets in submerged cultures, with little or no protease secretion. Cultural conditions favoring alkaline protease secretion have been optimized and a preliminary characterization of the enzyme is presented. Compatibility of the alkaline protease with commercial detergents as well as its potential application in recovering silver from spent photographic films have also been investigated.     

Overexpression of β-glucosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> for the production of highly purified aglycone isoflavones from soy flour

To produce aglycone isoflavones from soy flour, the β-glucosidase A gene (bglA) of Thermotoga maritima was overexpressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The K _m and V _max values of the purified BglA for pNPG were 0.43 mM and 323.6 U mg⁻¹, respectively, and those for salicin were 9.0 mM and 183.2 U mg⁻¹, respectively. The biochemical and kinetic characteristics of his-tagged BglA were found to be similar to those of BglA, except for the temperature stability and specific activity. Production of aglycone isoflavones from soy flour by BglA was examined by HPLC. For 3 h at 80°C, all the isoflavone glycosides approximated to the complete conversion into aglycone isoflavones, over seven times higher than that obtained from soy flour without BglA.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Production of the postharvest biocontrol agent Bacillus subtilis CPA-8 using low cost commercial products and by-products

The aim of this research was to identify a low cost medium based on commercial products and by-products that provided maximum Bacillus subtilis CPA-8 growth and maintained biocontrol efficacy. Low cost media combining economical nitrogen and carbon sources such as yeast extract, peptone, soy products, sucrose, maltose and molasses were tested. Tests were carried out in 250-ml flasks containing 50 ml of each tested medium. Maximum cell growth (>3 × 10⁹ CFU ml^?1) was obtained in defatted soy flour 44% combined with sucrose or molasses media. Second, CPA-8 production was scaled up in a 5-l fermenter and CPA-8 population dynamics, pH and oxygen consumption in the optimized medium (defatted soy flour 44% – molasses) was recorded. In these tests, there was a 5-h lag phase before growth, after which exponential growth occurred and maximum production was 3 × 10⁹ CFU ml^?1 after 20 h. Fruit trials with cells and cell free supernatants from CPA-8 grown in optimized medium maintained biocontrol efficacy against Monilinia fructicola on peaches, resulting in disease reductions up to 95%. CPA-8 populations survived in wounds on inoculated peaches, regardless of the culture media used. The results show that B. subtilis CPA-8 can be produced in a low cost medium combining inexpensive nitrogen and carbon sources (40 g l^?1 defatted soy flour 44%, 5 g l^?1 molasses plus mineral trace supplements) in shake flasks and a laboratory fermenter (5 l). The results could be used to provide a reliable basis for scaling up the fermentation process to an industrial level.     

Hydrolysis of soy isoflavone glycosides by recombinant β-glucosidase from hyperthermophile <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

A recombinant Thermotoga maritima β-glucosidase A (BglA) was purified to homogeneity for performing enzymatic hydrolysis of isoflavone glycosides from soy flour. The kinetic properties K _m, k _cat, and k _cat/K _m of BglA towards isoflavone glycosides, determined using high-performance liquid chromatography, confirmed the higher efficiency of BglA in hydrolyzing malonylglycosides than non-conjugated glycosides (daidzin and genistin). During hydrolysis of soy flour by BglA at 80°C, the isoflavone glycosides (soluble form) were extracted from soy flour (solid state) into the solution (liquid state) in thermal condition and converted to their aglycones (insoluble form), which mostly existed in the pellet to be separated from BglA in the reaction solution. The enzymatic hydrolysis in one-step and two-step approaches yielded 0.38 and 0.35 mg genistein and daidzein per gram of soy flour, respectively. The optimum conditions for conversion of isoflavone aglycones were 100 U per gram of soy flour, substrate concentration 25% (w/v), and incubation time 3 h for 80°C.     

Soybean bio-refinery platform: enzymatic process for production of soy protein concentrate,soy protein isolate and fermentable sugar syrup

Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process.     

Soy-based medium for ethanol production byEscherichia coli KO11

An optimized soy-based medium was developed for ethanol production byEscherichia coli KO11. The medium consists of mineral salts, vitamins, crude enzymatic hydrolysate of soy and fermentable sugar. Ethanol produced after 24 h was used as an endpoint in bioassays to optimize hydrolysate preparation. Although longer fermentation times were required with soy medium than with LB medium, similar final ethanol concentrations were achieved (44–45 g ethanol L^–1 from 100 g glucose L^–1). The cost of materials for soy medium (excluding sugar) was estimated to be $0.003 L^–1 broth, $0.006 L^–1 ethanol.     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus

An obligate alkalophilic Bacillus sphaericus strain, isolated from alkaline soils in the Himalaya, produced an extracellular protease which was optimally active at 50–55 °C and pH 10.5. The enzyme was stable in presence of 500 mg chlorine l^–1 and as a detergent additive. Its stability in presence of laundry detergents was comparable to that of commercial proteases. The gelatin layer in 25 g of used X-ray films was efficiently hydrolyzed within 12 min at 50 °C, pH 11.0 and 25 U protease/ml.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

Estimation of linear and mass attenuation coefficients of soy–lignin bonded Rhizophora spp. particleboard as a potential phantom material using caesium-137 and cobalt-60

In this study, linear and mass attenuation coefficients of fabricated particleboards intended for use as phantom material were estimated using ¹³⁷Cs and ⁶⁰Co radiation sources. Particleboards made of Rhizophora spp. wood trunk bonded with soy flour and lignin were fabricated at a target density of 1.0 g cm^?3, with and without gloss finish coating. Elemental composition of the particleboards was obtained by means of energy dispersive X-ray (EDX) spectroscopy. Experimental setups were simulated via the GATE Monte Carlo (MC) package, with particle histories of 1?×?10⁶–1?×?10⁷. Linear and mass attenuation coefficients obtained from measurements and GATE simulations were compared and discussed. The percentage differences between the measured and simulated linear and mass attenuation coefficients of the samples were reasonably small (2.05–4.88% for ¹³⁷Cs and 3.24–5.38% for ⁶⁰Co). It is shown that all the particleboards have the potential to be used as phantom materials as the attenuation coefficients measured were in good agreement with those of water (calculated with XCOM) and with those simulated with the GATE toolkit. The use of gloss finish coating also did not show any significant effect on the attenuation coefficient of the phantom material. Verification of experimental results via GATE simulations has been shown crucial in providing reliable data for energy transmission studies. Based on the results achieved in this study, it is concluded that the studied material—Rhizophora spp. wood trunk bonded with soy flour and lignin including gloss finish coating—can be used in radiation dosimetry studies.

     

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°C. The enzyme cleaved the oxidized insulin B chain initially at Leu¹⁵-Tyr¹⁶ and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH₄)₂SO₄-saturated acetate buffer (pH 5.0) as plank-like cyrstals.     

Nutrient-Enhanced Production of Remarkably High Concentrations of Ethanol by Saccharomyces bayanus through Soy Flour Supplementation

The supplementation of a simple medium with soy flour led to an increase in the specific growth rate and viable cell concentration of Saccharomyces bayanus during fermentation. Increasing the amount of soy flour led to an increase in the maximum number of viable yeast cells and the percentage of glucose fermented. It was possible in 64 h to reach 12.8% (wt/vol) ethanol by adding 4% soy flour (wt/vol) to a simple medium with 300 g of glucose per liter. The aqueous extract from soy flour was nearly as effective as whole-soy flour, whereas the lipidic fraction had no positive effect.     

Addendum to Issue 1 - ENZITEC 2012 Cheese whey and passion fruit rind flour as substrates for protease production by Bacillus sp. SMIA-2 strain isolated from Brazilian soil

Abstract

Application of wastes from the food processing industry as carbon sources in enzyme production processes reduces the cost of production, and also helps in solving problems of their disposal. In this work, we demonstrated that sweet cheese whey, in combination with passion fruit rind flour, can be successfully used for the production of protease by Bacillus sp. SMIA-2, opening perspectives for the use of these agricultural byproducts as novel and cost-effective culture media for the production of protease. The maximum production of the enzyme was observed in a sweet cheese whey-based culture medium preparation (0.5%, w/v) containing 0.25% (w/v) passion fruit rind flour and supplemented with different metal salts at an initial pH of 7.5–8.0, incubated at 50°C for 48 h. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 70°C. These characteristics presented by the protease produced by Bacillus sp. SMIA-2 could be very useful when thinking about biotechnological applications.     

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

Summary A chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.     

Utilization of Unfermented Cassava Flour for the Production of an Indigenous African Fermented Food, Agbelima

Summary Unfermented cassava flour, also called high quality cassava flour (HQCF) is used as a partial substitute for wheat flour in the baking industry. This work was carried out to produce an indigenous fermented cassava dough, agbelima, from high quality cassava flour in order to diversify uses for the flour. An isolate of each of the dominant species of lactic acid bacteria isolated from agbelima, was used to ferment reconstituted HQCF dough into agbelima, whilst pH changes, population on MRS, and the organoleptic quality were assessed. The antimicrobial properties of the fermenting samples against three enteric pathogens were also investigated. Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum and Leuconostoc mesenteroides were isolated at levels of 10¹⁰, 10⁹, 10⁹ and 10⁸ c.f.u. g⁻¹ respectively in agbelima. All isolates and a control sample, produced agbelima with pH of between 4.11 and 3.82 in 48 h, and ANOVA showed no significant difference between the pH of the samples at P ≤ 0.05. Spontaneous fermentation of the reconstituted dough was possible, because the flour was found to contain Lactobacillus plantarum at a level of 10⁶ c.f.u. g⁻¹ as well as the other species isolated from agbelima. Counts of Gram-positive catalase-negative rods and cocci on MRS in all the fermented samples were at levels of 10⁸ to 10⁹ c.f.u. g⁻¹, with ANOVA showing no significant difference between the populations at P ≤ 0.05. Three enteric pathogens, Salmonella typhimurium 9, Echerichia coli D2188 and Vibrio cholerae C-230, inoculated into the samples at 10⁶ to 10⁷ c.f.u. g⁻¹ at the start of fermentation, could not be detected in 10 g of any of the samples at the end of 48 h fermentation. A taste panel preferred banku - a stiff porridge made from a combination of fermented maize dough and fermented cassava dough – prepared with fermented HQCF dough to banku prepared with a market sample of agbelima or reconstituted agbelima flour. Processing the highly perishable cassava roots into high quality cassava flour, therefore, offers a means for preserving cassava, which can subsequently be used for both industrial and traditional purposes.     

A high salt tolerant neutral protease from Aspergillus Oryzae: Purification, characterization and kinetic properties

A neutral high salt tolerant protease from Aspergillus oryzae CICIM F0899 which could be used for soy sauce production and other relevant applications under high-salt conditions was purified to homogeneity through ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography with overall recovery of 2%. Its molecular weight was estimated to be 50 kDa by SDS-PAGE. The optimum pH and temperature for activity of the extracellular protease of A. oryzae CICIM F0899 were shown to be between 7.0–9.0, and 50°C, respectively. The protease behaved high salt tolerance in 18% NaCl and retained 72% of initial activity after 14 days, indicating the high stability. The enzyme activity was inhibited by metal ions such as Al³⁺ and Ag⁺, and slightly activated by Mn²⁺ and Cu²⁺. A kinetic model incorporating the Debye-Hückel limiting law was proposed for A. oryzae CICIM F0899 protease hydrolysis of casein at ionic strength NaCl from 0.10 to 3.18 M. It was found that, with the higher ionic strength, the Michaelis constant K _m of the protease monotonically increased while the turnover number k _cat decreased in accordance with first order kinetic model. The high-salt tolerant protease has been demonstrated to be promising for the soy sauce production process.