Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation

Cutinases comprise a family of esterases with broad hydrolytic activity for chain and pendant ester groups. This work aimed to identify and improve an efficient cutinase for cellulose acetate (CA) deacetylation. The development of a mild method for CA fiber surface deacetylation will result in improved surface hydrophilicity and reactivity while, when combined with cellulases, a route to the full recycling of CA to acetate and glucose. In this study, the comparative CA deacetylation activity of four homologous wild‐type (wt) fungal cutinases from Aspergillus oryzae (AoC), Thiellavia terrestris (TtC), Fusarium solani (FsC), and Humicola insolens (HiC) was determined by analysis of CA deacetylation kinetics. wt‐HiC had the highest catalytic efficiency (≈32 [cm² L^‐1]^‐1 h^‐1). Comparison of wt‐cutinase catalytic constants revealed that differences in catalytic efficiency are primarily due to corresponding variations in corresponding substrate binding constants. Docking studies with model tetrameric substrates also revealed structural origins for differential substrate binding amongst these cutinases. Comparative docking studies of HiC point mutations led to the identification of two important rationales for engineering cutinases for CA deacetylation: (i) create a tight but not too closed binding groove, (ii) allow for hydrogen bonding in the extended region around the active site. Rationally designed HiC with amino acid substitutions I36S, predicted to hydrogen bond to CA, combined with F70A, predicted to remove steric constraints, showed a two‐fold improvement in catalytic efficiency. Continued cutinase optimization guided by a detailed understanding of structure‐activity relationships, as demonstrated here, will be an important tool to developing practical cutinases for commercial green chemistry technologies.     

Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride

We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (muHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol(-1) and -2.6 kcal mol(-1) for GdnHCl and AOT, respectively. Hence, the AOT denatured state (D(AOT)) is less destabilised than the GdnHCl denatured state (D(GdnHCl)), relative to the native state in water. Far-UV CD spectroscopy revealed that D(AOT) retains some secondary structure, while D(GdnHCl) is essentially unstructured. Similarly, fluorescence data suggest that D(AOT) is more compact than D(GdnHCl). Activity measurements reveal that both D(AOT) and D(GdnHCl) are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of D(AOT) in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of D(AOT) in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data.     

Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum

A GH3 β-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated temperatures with no loss in activity after 24 h of incubation at 60°C at pH 4–6, and the BGL was shown to have significantly higher stability at these conditions in comparison to Novozym 188 and to other fungal GH3 BGLs reported in the literature. The BGL had significant lower affinity for cellobiose compared with the artificial substrate para-nitrophenyl-β-d-glucopyranoside (pNP-Glc) and further, pronounced substrate inhibition using pNP-Glc. Kinetic studies demonstrated the high importance of using cellobiose as substrate and glucose as inhibitor to describe the inhibition kinetics of BGL taking place during cellulose hydrolysis. A novel assay was developed to characterize this glucose inhibition on cellobiose hydrolysis. The assay uses labelled glucose-¹³C₆ as inhibitor and subsequent mass spectrometry analysis to quantify the hydrolysis rates.     

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl₂. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0–6.6. Na⁺, K⁺, Ca²⁺, Mg²⁺ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe³⁺. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K _m and V _max values against salicin as substrate were 0.035 mM and 1.7215 μmol min⁻¹, respectively.     

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (T_g) of PET, that is, 70°C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6°C improvement in the thermal unfolding temperature (T_m) and a 10‐fold increase in the half‐life of the enzyme activity at 60°C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the T_m of the global structure. Proteins 2016; 84:60–72. © 2015 Wiley Periodicals, Inc.     

Cloning,purification, and characterization of β-galactosidase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> DSM 13

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K _m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na⁺ and K⁺ in the concentration range of 1–100 mM as well as the divalent metal cations Mg²⁺, Mn²⁺, and Ca²⁺ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.     

Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.     

α-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH

An a-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50°C. The K _m values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl α-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.     

Biochemical and molecular characterization of a cellobiohydrolase from <Emphasis Type="Italic">Trametes versicolor</Emphasis>

A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and temperature optima were 5.0 and 40°C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and at temperatures lower than 50°C. The kinetic parameters with the substrate p-nitrophenyl β-d-cellobioside (pNPC) were 0.58 mM and 1.0 μmol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a catalyzes the hydrolysis of pNPC, filter paper, β-glucan, and avicel to varying extents, but no detectable hydrolysis was observed when using the substrates carboxymethylcellulose, laminarin and pNPG.     

A xylanase with high pH stability from Streptomyces sp. S27 and its carbohydrate-binding module with/without linker-region-truncated versions

A xylanase gene, xynAS27, was isolated from a genomic library of Streptomyces sp. S27. The full-length gene consists of 1,434 bp and encodes 477 amino acids, including a putative signal peptide of 41 residues at its N-terminus. The mature xylanase comprises two functional domains, a family 10 glycoside hydrolase, and a family 13 carbohydrate-binding module (CBM), which were joined by a short Gly/Pro-rich linker region. The intact, the CBM-truncated and the CBM-linker-truncated versions of the mature proteins were expressed in Escherichia coli BL21 (DE3), purified to electrophoretic homogeneity and subsequently characterized. XynAS27 showed high pH stability over the pH range 2.2–12.0. XynAS27 may be a compelling tool for the food industry because it generates xylobiose (85% w/w) as the main product of xylan hydrolysis. The truncated versions showed less pH and thermal stability, and less affinity and hydrolytic activity to insoluble substrate than the intact one. These results indicate that the CBM of XynAS27 plays a key role in the hydrolysis of insoluble substrate, and the CBM and linker region are also important for the enzyme stability, and the linker region contributes more.     

Entrapment of β-galactosidase in polyvinylalcohol hydrogel

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g⁻¹) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.     

Isolation and properties of extracellular β-xylosidases from fungi <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> and <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Homogeneous β-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5–4.0), and temperature activity optimum was 70°C for the β-xylosidase of A. japonicus and 60°C for the β-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl β-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that β-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while β-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was found. The β-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with α-L-arabinofuranosidase and endoxylanase upon hydrolysis of arabinoxylan. Addition of β-xylosidases increased efficiency of hydrolysis of plant raw materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own β-xylosidase.     

Modulation of pH Sensitivity of Surface Charge and Aggregation Stability of Protein-Coated Lipid Droplets by Chitosan Addition

The impact of a cationic polyelectrolyte on the pH sensitivity of the electrical charge and aggregation stability of protein-coated lipid droplets was examined. One percent (w/w) corn oil-in-water emulsions containing lipid droplets coated by β-lactoglobulin [0.05% (w/w) β-Lg, 10 mM acetate buffer, pH 3] were prepared in the absence (“primary” emulsions) and presence (“secondary” emulsions) of chitosan (0 to 0.05 wt%). The pH (3 to 8) of these emulsions was adjusted, and the particle charge, particle size, creaming stability, and microstructure were measured. Chitosan adsorbed to the β-Lg-coated droplets from pH 4.5 to 7.5, which was attributed to electrostatic attraction between the cationic polyelectrolyte and anionic patches on the droplet surfaces. Droplets coated by β-Lg–chitosan had better stability to flocculation than those coated by β-Lg alone around the isoelectric point of the adsorbed proteins (pH 4.5 to 5.5), which was attributed to increased electrostatic and steric repulsion between the droplets. We have shown that chitosan may be used to modulate the electrical characteristics and stability of protein-coated lipid droplets, which may be useful in the design and formation of delivery systems for use in the food, pharmaceutical, and other industries.     

Production of Cellulases by Aspergillus fumigatus and Characterization of One β-Glucosidase

Aspergillus fumigatus produces substantial extracellular cellulases on several cellulosic substrates including simple sugars. Low glucose potentiates enzyme production, but most cellulose-induced cellulases are repressed by high glucose. As production of cellulase in a wide substrate range is unusual, the cellulolytic complex of this thermophilic fungus was investigated. A β-glucosidase was separated by gel filtration and ion-exchange chromatography. It migrated in native polyacrylamide gel as a single protein (130 kDa), which split under denaturing conditions into two smaller proteins having molecular masses of 90 kDa and 45 kDa. However, only the 90-kDa protein was active. Conventional chromatographic procedures were unsuccessful for the separation of these two proteins. Therefore, the 130-kDa protein was studied for its kinetic properties. It hydrolyzed p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose, but not β-glucans, laminarin, and p-nitrophenyl-β-D-xilopyranoside. The optimal pH and temperature of p-NPG and cellobiose hydrolysis were 5.0 and 4.0, and 65°C and 60°C, respectively. The K _m values, determined for cellobiose and p-NPG of hydrolysis, were 0.075 mM and 1.36 mM, respectively. Glucose competitively inhibited the hydrolysis of p-NPG. The K_i was 3.5 mM.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

PepJ is a new extracellular proteinase of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 °C, respectively. The enzyme was stable at pH 3.5–10.5 and was still active at 95 °C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.     

Purification and characterization studies of a thermostable β-xylanase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis>

This study presents data on the production, purification, and properties of a thermostable β-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0–5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K _m values (mg ml⁻¹) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50°C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.     

Homologue expression of a β-xylosidase from native <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. β-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-β-xylanase activity. This work reports the partial characterization of a purified β-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding β-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. β-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein⁻¹ at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol⁻¹, and at 50°C optimum stability was shown at pH 4.0–5.0. β-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), β-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn²⁺. Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.     

Degradation of Nucleic Acids and Their Related Compounds by Microbial Enzymes

The optimum pH of the DNA-depolymerase produced by Aspergillus quercinus was found to be about 8.5 and maximal formation of the enzyme in the culture medium was observed at the 96th hour. The culture filtrate of Aspergillus quercinus hydrolyzed DNA into 5′-deoxy-mononucleotides at a pH range higher than 6.0. Each deoxymononucleotide was isolated as crystals in good yield from an enzymatic digest of DNA and characterized spectfophotometri-cally, enzymatically and by determination of its nitrogen and phosphorus composition. 5′-Deoxyinosinic acid was obtained by hydrolysis of DNA with Streptomyces aureus. 5′-Deoxyribo-tides of hypoxanthine and guanine possessed an attractive taste very similar to that of 5′-ino-sinic and 5′-guanylic acids.