Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Purification and Characterization of a Novel Thermostable Xylose Isomerase from <Emphasis Type="BoldItalic">Opuntia vulgaris</Emphasis> Mill

Thermophilic xylose isomerase from the xerophytic eukaryote Opuntia vulgaris can serve as a good alternate source of enzyme for use in the production of high fructose corn syrup. The existence of two temperature stable isoforms having optimal activity at temperatures 70 °C (T₇₀) and 90 °C (T₉₀), respectively, is reported here. These isoforms were purified to homogeneity using column chromatography and SDS-polyacrylamide gel electrophoretic techniques. Only the T₉₀ isoform was subjected to full biochemical characterization thereafter. The purified T₉₀ isoform was capable of converting glucose to fructose with high efficiency under the assay conditions. The enzyme at pH 7.5 exhibited a preference to yield the forward isomerization reaction. The melting temperature of the native enzyme was determined to be 90 °C employing differential scanning colorimetery. Thermostability of the enzyme protein was established through temperature-related denaturation kinetic studies. It is suggested that the thermostability and the wide pH activity of this eukaryotic enzyme will make it an advantageous and dependable alternate source of catalytic activity for protected use in the high fructose corn syrup sweetener industry.     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K _m and V _max values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 μmol/min/mg, respectively. PG was found to have temperature optimum at 65 °C and was totally stable at 45 °C for 90 min. Half-life at 55 °C was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na⁺¹, K⁺¹, and Co⁺² (1 mM) and Cu⁺² (1 and 10 mM).     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

<Emphasis Type="Italic">Rhizopus stolonifer</Emphasis> LAU 07: a novel source of fructosyltransferase

Increasing awareness of the importance of fructooligosaccharides (FOS) as ingredients of functional foods has led to intensive search of new sources of fructosyltransferases (FTase), enzymes responsible for the conversion of sucrose to fructooligosaccharides. A local strain of Rhizopus stolonifer isolated from spoilt orange fruit with high fructosyltransferase activity (U _t) of 12.31–45.70 U mL⁻¹ during a fermentation period of 24–120 h is herein reported. It showed low hydrolytic activity (U _h) in the range of 0.86–1.78 U mL⁻¹ during the same period. FOS yield of 34 % (1-kestose, GF₂, nystose, GF₃) was produced by FTase obtained from a 72 h-old culture using 60 g of sucrose per 100 mL of the substrate. When the isolate was grown in a defined submerged medium, its pH dropped sharply from the intial value of 5.5 to 1.0 within 24 h, and this value was maintained throughout the fermentation. The biomass content ranged from 8.8 g L⁻¹ at 24 h of fermentation to reach the maximum of 10 g L⁻¹ at 72 h. It was reduced to 5.6 g L⁻¹ at the end of 120 h of fermentation. This report represents the first reference to a strain of Rhizopus as a source of FTase for the production of FOS. The high U _t/U _h ratio shown by this isolate indicates that it may be a good strain for the industrial and commercial production of FOS. However, there is a need of further optimization of the bioprocess to increase the conversion efficiency of sucrose to FOS by the enzyme.     

Production of the Enzyme Cyclodextringlycosyltransferase from Bacillus firmus Alkalophilic

Different culture media have been testedfor the production of the enzyme CGTase (cyclodextringlycosyltransferase) from Bacillus firmus (strain #37). The concentration of different carbon and nitrogen sources have been varied and the enzyme activity, cell concentration, reducing sugars, total reducing sugars, soluble protein and pH have been followed during cultivation. Results indicate that higher concentrations of yeast extract and polypeptone lead to increased synthesis of CGTase, whereas when starch is substituted by glucose there is a drastic inhibition of CGTase production.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.     

Effects of Carbon and Nitrogen Sources and Oxygenation on the Production of Inulinase by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Cultivations of Kluyveromyces marxianus var. bulgaricus ATCC 16045 were performed on both minimal and complex media using different carbon and nitrogen sources either in the presence or absence of aeration. The results collected were worked out and compared so as to provide a useful contribution to the optimization of inulinase production. Kinetics of extracellular inulinase release were similar on glucose, fructose, and sucrose. Inulinase was detected at basal level since the beginning of batch runs on these three carbon sources and overproduced after their depletion. The highest inulinase activity in minimal medium containing 10 g/l sucrose (6.4 IU/ml) was obtained at an initial (NH₄)₂SO₄ concentration of 5 g/l, whereas it was reduced to about one fourth of this value and detected only at the beginning under nitrogen-limited conditions. The best sucrose concentrations for the enzyme production were 30 and 20 g/l in minimal and complex media, yielding 15.4 and 208 IU/ml, respectively. In general, the enzyme activity was much higher in complex than in minimal medium under all conditions. O₂-enriched air neither improved inulinase production nor prevented ethanol formation.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Design variations of a polymer–enzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependence

The glucose sensitivity and oxygen dependence of a variety of implantable biosensors based on glucose oxidase (GOx), incorporating an electrosynthesized poly-o-phenylenediamine (PPD) permselective barrier on 125-μm diameter Pt disks (Pt_D) and cylinders (Pt_C, 1-mm length), were measured and compared. Full glucose calibrations and experimental monitoring of solution oxygen concentration allowed us to determine apparent Michaelis–Menten parameters for glucose and oxygen. In the linear region of glucose response, the most sensitive biosensor design studied was Pt_D/PPD/GOx (enzyme deposited over polymer) that was 20 times more sensitive than the more widely used Pt_C/GOx/PPD (enzyme immobilized before polymer deposition) configuration. The oxygen dependence, quantified as K_M(O₂), of both active and less active designs was surprisingly similar, a finding that could be rationalized in terms of an increase in K_M(G) with increased enzyme loading. The Pt_D/PPD/GOx design will now enable us to explore glucose concentration dynamics in smaller and layered brain regions with good sensitivity and minimal interference from fluctuations in tissue pO₂.     

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.     

Effect of Different Carbon Sources on Cellulase Production by Marine Strain Microbulbifer hydrolyticus IRE-31-192

Cellulase is an important enzyme that can be used to breakdown lignocellulose into glucose. Microbulbifer hydrolyticus IRE-31(ATCC 700072) is a kind of marine bacterium, which could grow in high salinity medium and has fast-strong growth ability. In this study, a novel strain was screened from Microbulbifer hydrolyticus IRE-31 through mutations to produce cellulase. The effect of different carbon sources on the growth as well as on the production of cellulase of the new strain was studied. Carboxymethyl-cellulase (CMCase) activity selected to represent cellulase was proven to be effectively promoted while xylose, galactose, and melibiose as well as glucose were used as carbon sources. When xylose and glucose were chosen to be further investigated, 472.57 U/L and 266.01 U/L CMCase activity were obtained from 30 g/L glucose and 10 g/L xylose, respectively. These results clarified the effect of different carbon sources on the production of cellulase, which laid a good foundation for the further research in the production of cellulase by marine bacteria.

     

Effect of temperature on growth of psychrophilic and psychrotrophic members of Rhodotorula aurantiaca

The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate (μ_max), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum cell production was observed at about 0°C, and cell production decreased when temperature increased. The μ_max values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.     

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg_prot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg_prot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.