Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation

Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70 °C, with a 3.0 KJ mol⁻¹ increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6 U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3 U/mL, followed by cassava starch and corn starch with 5.9 and 3.0 U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose–Sepharose affinity chromatography; the estimated molecular mass was 100 kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65 °C.     

Horticultural Waste as the Substrate for Cellulase and Hemicellulase Production by Trichoderma reesei Under Solid-State Fermentation

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions. This enzyme mixture contained FPase (15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase (61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM). The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM. The potential of the crude enzyme complex produced was demonstrated by the hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The performance of the crude enzyme complex was better than the commercial enzyme blend.     

Comparison of citric acid production by solid-state fermentation in flask, column, tray, and drum bioreactors

Studies were conducted to evaluate citric acid production by solid-state fermentation (SSF) using cassava bagasse as substrate employing a fungal culture of Aspergillus niger LPB 21 at laboratory and semipilot scale. Optimization of the process parameters temperature, pH, initial humidity, aeration, and nutritive composition was conducted in flasks and column fermentors. The results showed that thermal treatment of cassava bagasse enhanced fungal fermentation efficacy, resulting in 220 g of citric acid/kg of dry cassava bagasse with only treated cassava bagasse as substrate. The results obtained from the factorial experimental design in a column bioreactor showed that an aeration rate of 60 mL/min (3 mL/[g·min]) and 60% initial humidity were optimum, resulting in 265.7 g/kg of dry cassava bagasse citric acid production. This was almost 1.6 times higher than the quantities produced under unoptimized conditions (167.4 g of citric acid/kg of dry cassava bagasse). The defined parameters were transferred to semipilot scale, which showed high promise for large-scale citric acid production by SSF with cassava bagasse. Respirometry assays were carried out in order to follow indirectly the biomass evolution of the process. Citric acid production reached 220, 309, 263, and 269 g/kg of dry cassava bagasse in Erlenmeyer flasks, column fermentors, a tray bioreactor, and a horizontal drum bioreactor, respectively.     

High-Yield Hypocrellin A Production in Solid-State Fermentation by <Emphasis Type="Italic">Shiraia</Emphasis> sp. SUPER-H168

Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 × 10⁶ spores, substrate particle size 0.8–1 mm, initial moisture content 50%, and temperature 30 °C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO₃ were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO₃ were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.     

Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of <Emphasis Type="Italic">Bacillus cereus</Emphasis> C<Subscript>7</Subscript>

A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.     

Solid-State fermentation of phytase from cassava dregs

Phytases produced by numerous microorganisms and plants degrade phytic acid that has chelated with metal ions in food and feed. It is important to study phytase for the role of metal ions in nutrition of animals and humans as wellas in the reduction of organic phosphate content of a queous environment. This article reports on solid-state fermentation of phytase from a new substrate of cassava dregs. Large quantities of cassava dregs are produced in tropical areas as a by product of cassava starch processing. Protein and inorganic salts were found to be low in cassava dregs. Cassava dregs could be employed for phytase synthesis after the addition of a nitrogen source and mineral salts. Ammonium nitrate was the best nitrogen source among the nitrogen sources investigated, including beef extract, yeast extract, urea, ammonium nitrate, sodium nitrate, and ammonium sulfate. Sodium dodecyl sulfate promoted phytase production from cassava dregs. A maximum phytase yield of 6.73 U/g of dry mass was obtained. The obtained phytase was stable at feed-processing temperature, since 70% of initial enzyme activity was maintained after 30 min of treatment at 75°C.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Utilization of Horticultural Waste for Laccase Production by <Emphasis Type="Italic">Trametes versicolor</Emphasis> Under Solid-State Fermentation

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.     

New Thermostable Amylase from <Emphasis Type="Italic">Bacillus cohnii</Emphasis> US147 with a Broad pH Applicability

A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed K _m and V _max values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 °C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 °C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO₃, 1 mM H₂O_2, 1 mM Zn⁺², and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca ²⁺ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

Water Hyacinth as Carbon Source for the Production of Cellulase by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.     

Gene Cloning,Heterologous Expression,and Characterization of a High Maltose-Producing α-Amylase of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4–6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5–6.5 and temperatures below 50 °C. Purified RoAmy had a K _m and V _max of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu²⁺ and 5 mM Fe²⁺, whereas 5 mM Ca²⁺ showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K _m = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.     

Biochemical Characterization of Raw-starch-digesting Alpha Amylase Purified from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

Alpha amylase (E.C. 3.2.1.1) of Bacillus amyloliquefaciens produced by submerged fermentation was purified to near homogeneity by ion exchange chromatography. Through the process 38.6-fold increase in purity with a specific activity of 72 U/mg proteins was obtained. The apparent molecular weight of the purified enzyme was found to be 58 kDa by SDS-PAGE. The enzyme was relatively stable between pH 5.0–8.0 and temperature between 50 and 60°C. The enzyme did not show any obligate requirement of metal ions but Ca²⁺ and Cu²⁺ enhanced the enzyme activity marginally and the thermostability was enhanced in the presence of Ca²⁺ ions. The purified enzyme exhibited maximal substrate specificity for amylose and efficiency in digesting various raw starches. The K _m and V _max of the enzyme was determined using both amylose and soluble starch as substrate. The analysis of the hydrolyzed products of soluble starch by thin layer chromatography showed the yield of maltosaccharides after 6 h of hydrolysis.     

Butanol Production from Cane Molasses by <Emphasis Type="Italic">Clostridium saccharobutylicum</Emphasis> DSM 13864: Batch and Semicontinuous Fermentation

Clostridium acetobutylicum strains used in most Chinese ABE (acetone–butanol–ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD₆₆₀, respectively. The average batch fermentation time (stage I and II) was reduced to 21−25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate.     

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.     

Characterization and performance of immobilized amylase and cellulase

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.