Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase

pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60°C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.     

Solid-state fermentation for production of phytase by Rhizopus oligosporus

Solid-state fermentation of coconut oil cake has been carried out with Rhizopus oligosporus for the production of phytase. Phytase is used commercially in the animal feed industry to improve animal performance because there is a substantial and growing interest among swine and poultry producers in the application of phytase to improve the nutritional quality in animal feeds. Demonstrated benefits include improved feed yield ratios and reduction in the environmental costs associated with the disposal of animal wastes. We report the production of extracellular phytase by R. oligosporus under solid-state fermentation using coconut oil cake as substrate. Maximal enzyme production (14.29 U/g of dry substrate) occurred at pH 5.3, 30°C, and 54.5% moisture content after 96 h of incubation. The addition of extra nutrients to the substrate resulted in inhibition of product formation. The results indicate the scope for production of phytase using coconut oil cake as solid substrate without additional nutrients.     

1,2-Diazole and 2,2,2-Trifluoroethanol and Their Regulatory Effects on Ethanol and Lactic Acid Formation in the Living Culture of Rhizopus oryzae

In heterofermentation of Rhizopus oryzae, ethanol is the major byproduct which reduces the production of a desired product, an optically pure l-lactic acid. To improve lactic acid production, regulating the alcohol fermentative pathway to limit ethanol production has been done by various techniques. In vitro study on alcohol dehydrogenase (ADH) inhibition in several organisms showed that 1,2-diazole and 2,2,2-trifluoroethanol were competitively bound at the active sites that eventually limited ethanol production. In this study, 1,2-diazole and 2,2,2-trifluoroethanol were present during fermentation of R. oryzae. It was found that both 1,2-diazole and 2,2,2-trifluoroethanol not only strongly affected ethanol formation but they also indirectly regulated lactate production as observed by the decreasing affinity for glucose flux toward lactate and ethanol production. The increase in both ethanol and lactate formation rates revealed 1,2-diazole and 2,2,2-trifluoroethanol not only regulated the reversible redox reaction by ADH, but they also caused the dynamic change in the conversion of all metabolites in the living R. oryzae in order to maintain the balanced flux for cellular growth and maintenance.     

Genome Mining for New α-Amylase and Glucoamylase Encoding Sequences and High Level Expression of a Glucoamylase from Talaromyces stipitatus for Potential Raw Starch Hydrolysis

Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD₆₀₀?>?200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ～8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71–87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.     

Production of Fumaric Acid by Bioconversion of Corncob Hydrolytes Using an Improved <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Strain

The use of microorganism fermentation for production of fumaric acid (FA), which is widely used in food, medicine, and other fields, can provide technical support for the FA industry. In this study, we aimed to increase the titer of FA production by using an improved Rhizopus oryzae WHT5, which was domesticated to obtain a furfural-resistant strain in corncob hydrolytes. The metabolic pathways and metabolic network of this strain were investigated, and the related enzymes and metabolic flux were analyzed. Metabolic pathway analysis showed that the R. oryzae WHT5 strain produced FA mainly through two pathways. One occurred in the cytoplasm and the other was a mitochondrial pathway. The key parameters of the fermentation process were analyzed. The FA titer was 49.05 g/L from corncob hydrolytes using R. oryzae WHT5 in a 7-L bioreactor. The use of a furfural-resistant strain developed through domestication effectively increased the titer of FA. This capacity of the microorganisms to produce high amounts of FA by bioconverting corncob hydrolyte can be further applied for industrial production of FA.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

Manipulating Pyruvate Decarboxylase by Addition of Enzyme Regulators during Fermentation of Rhizopus oryzae to Enhance Lactic Acid Production

Ethanol was found as the major by-product in lactate fermentation by Rhizopus oryzae. Several methods have been conducted in order to limit ethanol formation, thus increasing the lactate yield. The direct way to suppress ethanol production can be done by inhibition of the responsible enzymes in the related pathway. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are responsible for ethanol production in R. oryzae. Shunting the ethanol production pathway by targeting at PDC was attempted in this study. Three compounds including 4-methylpyrazole, glyoxylic acid, and 3-hydroxypyruvate with the in vitro reversible inhibitory effect on PDC were selected from the literature and were used to regulate the living cell of R. oryzae during the fermentation. The results show that 0.1 mM 4-methylpyrazole of which the structure resembled a thiazolium ring in thiamine diphosphate, PDC cofactor, and 1.0 μm 3-hydroxypyruvate, pyruvate analog, effectively hampered ethanol production. Further observation on the enzyme expression indicated that these two regulators not only targeted PDC but also caused changes in ADH and lactate dehydrogenase (LDH) activities. This was perhaps due to the living cell of R. oryzae that responded to the presence of the regulators to balance the pyruvate flux and subsequently maintain its metabolic activities.     

In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in <Emphasis Type="Italic">Rhizopus Oryzae</Emphasis> to Improve <Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid Fermentation

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.     

Simultaneous Saccharification of Inulin and Starch Using Commercial Glucoamylase and the Subsequent Bioconversion to High Titer Sorbitol and Gluconic Acid

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.     

Optimization of L-Lactic acid production from glucose by Rhizopus oryzae ATCC 52311

The effect of nutrients on L(+)-lactic acid production from glucose was investigated using Rhizopus oryzae ATCC 523 11. From the shake-flask experiments, the optimal medium composition was defined for improved lactic-acid production. In order to enhance lactic-acid production rate and product yield, controlled aeration in a bubble column was conducted under optimal conditions. Results showed a maximum lactic-acid production rate of 2.58 g/L/h was obtained with an initial glucose concentration of 94 g/L. Finallactic-acid concentration of 83 g/L was achieved after 32 h of fermentation with a weight of 0.88 glactic acid/g glucose consumed.     

Enhanced Fumaric Acid Production from Brewery Wastewater and Insight into the Morphology of Rhizopus oryzae 1526

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5 % (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465?±?0.04 mm), the highest concentration of fumaric acid achieved was 31.3?±?2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.     

Use of Mesophilic Fungal Amylases Produced by Solid-state Fermentation in the Cold Hydrolysis of Raw Babassu Cake Starch

Amylases constitute one of the most important groups of industrial enzymes, presenting several applications, such as in the food, textile, and ethanol manufacturing. In this work, a starchy residue from the Brazilian agroindustry, namely babassu cake, was used for the production of amylases by solid-state fermentation and for obtaining sugar hydrolysates, which can be used as building blocks for future bioconversions. Eight filamentous fungi from the genera Aspergillus and Penicillium were screened. Regarding amylase production, A. awamori strains showed well-balanced endoamylase and exoamylase activities, while A. wentii produced an amylolytic complex much richer in the endo-acting enzymes. Simultaneous liquefaction and saccharification using the crude enzyme extracts from the four most promising fungal strains was then investigated applying DOE techniques. The highest total reducing sugar content (24.70 g L^?1) was obtained by the crude extract from A. awamori IOC-3914, corresponding to a hydrolysis yield of 52% of total starch in the cake, while A. awamori IOC-3915 produced the most appropriate extract in terms of glucose release (maximum of 5.52 g L^?1). Multivariate analysis of the DOE studies indicated that these extracts showed their best performance at 50–57 °C under acid conditions (pH 3.6–4.5), but were also able to act satisfactorily under milder conditions (36 °C and pH 5.0), when TRS and glucose released were about 65% of the maximum values obtained. These data confirm the high potential of the enzyme extracts under study for cold hydrolysis of starch.     

Catalytic Properties of Two <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> 99-880 Glucoamylase Enzymes Without Starch Binding Domains Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k _cat and K _m for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K _m ^maltose . AmyD has a 3-fold higher K _m and lower k _cat for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K _i for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.     

On-line FT-Raman spectroscopic monitoring of starch gelatinisation and enzyme catalysed starch hydrolysis

The hydrolysis of starch by α-amylase and amyloglucosidase and the kinetics of these technically important reactions were investigated by FT-Raman spectroscopy. During the technical starch hydrolysis process, three reactions proceed one after the other but partly in parallel: the gelatinisation, the hydrolysis of starch to dextrin (liquefaction) by α-amylase, and the hydrolysis of dextrin to glucose (saccharification) by glucoamylase. These three reactions were studied separately. Potato starch or dextrin in concentrations of 50 g/l were suspended or dissolved in water, the reaction chamber was placed in a heated water bath, and defined, varied amounts of enzymes in solution were added. The reactions were monitored on-line in a bypass loop. The greatest spectral changes were observed due to the swelling and gelatinisation of starch. The liquefaction of starch to dextrin was started with the addition of α-amylase at a temperature of 80°C and the spectral changes were monitored. In a similar way, a solution/suspension of dextrin was used to investigate the reaction of glucoamylase at 50°C. Both enzymatic reactions were performed at four different enzyme activities. All reactions showed distinctive spectral changes, which can, in principle, be evaluated for the determination of the degree of starch hydrolysis. As a Nd:YAG laser at 1064 nm was used, fluorescence excitation was not observed despite the use of crude, technical-grade enzymes. The findings demonstrate the potential use of Raman spectroscopy in monitoring and control of technical enzyme reactions.     

Development of Batch and Continuous Processes on Biodiesel Production in a Packed-Bed Reactor by a Mixture of Immobilized Candida rugosa and Rhizopus oryzae Lipases

In this study, transesterification and esterification were investigated in batch and continuous process using immobilized Candida rugosa and Rhizopus oryzae lipases. In the case of batch process, stepwise reaction method was investigated to prevent the lipase deactivation. Reaction conditions were as follows: temperature, 45 °C; agitation speed, 250 rpm; enzyme concentration, 20%; and water contents 10%. And then, conversion yield was 98.33% at 4 h. In the case of continuous process, circulation and long-term continuous system were investigated for development of efficient mass transfer system. Optimal reaction conditions were as follows: temperature, 45 °C; flow rate, 0.8 mL/min; and water contents, 10%. And then, conversion yield of biodiesel was 97.98% at 3 h. Especially, the maximum conversion yield using a mixture of immobilized lipases exceeded over 90% for 108 h in long-term continuous system under optimal reaction conditions (45 °C; flow rate, 0.8 mL/min; and water contents, 10%). These results should help in determining the best method for the biodiesel production and improving the design and operation of large scale by enzymatic systems.     

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

A HPLC method for specific determination ofα-amylase and glucoamylase in complex enzymatic preparations

Summary In the hydrolysis of soluble starch by mixtures of α-amylase and glucoamylase, the ratios maltose/glucose and maltoriose/glucose linearly depend, over a wide range, on the relation between both enzymes and are independent on the activity level of the enzymatic preparation. HPLC determination of hydrolysis products (glucose, maltose and maltotriose) of soluble starch by mixtures of these enzymes, after incubation under controlled conditions, is a rapid method for the evaluation of the relative levels of each enzyme in the mixtures. The method, first developed using pure commercial amylases, is applied, with consistent results, to cell free media ofAspergillus niger cultures on a glycogen-rich effluent.     

Characterization of a Neutral and Thermostable Glucoamylase from the Thermophilic Mold Thermomucor indicae-seudaticae: Activity, Stability, and Structural Correlation

Glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae was purified by anion exchange and gel filtration chromatographic techniques using a fast protein liquid chromatographic system. The structure and thermal stability of this unique ‘thermostable and neutral glucoamylase’ were analyzed by circular dichroism (CD). T. indicae-seudaticae glucoamylase (TGA) contained typical aromatic amino acid (tryptophan/tyrosine) fingerprints in its tertiary structure. Analysis of the far-UV CD spectrum at pH 7.0 and 25 °C revealed the presence of 45% α-helix, 43% β-sheet, and 12% remaining structures. The α-helix content was highest at pH 7.0, where glucoamylase is optimally active. This observation points towards the possible (α/α)₆ barrel catalytic domain in TGA, as reported in microbial glucoamylases. Thermal denaturation curves of the pure protein at different pH values revealed maximum stability at pH 7.0, where no change in the secondary structure was observed upon heating in the temperature range between 20 °C and 60 °C. The observed midpoint of thermal denaturation (T _m) of glucoamylase at pH 7.0 was 67.1 °C, which decreased on either sides of this pH. Thermostability of TGA enhanced in the presence of starch (0.1%) as no transition curve was obtained in the temperature range between 20 °C and 85 °C. The only product of TGA action on starch was glucose, and it did not exhibit transglycosylation activity even at 40% glucose that can also be considered as an advantage during starch saccharification.     

Mycelial pellet formation by Rhizopus oryzae ATCC 20344

Factors in a cultivation medium affecting fungal growth morphology and funmaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted, the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.