Feruloyl esterase production by Aspergillus terreus CECT 2808 and subsequent application to enzymatic hydrolysis

Ferulic acid esterases (FAE) were produced by Aspergillus terreus CECT 2808 from vine trimming shoots (VTS) and corn cob. Later, the fungal extracts thus obtained were used to enzymatically release ferulic acid (FA) from both substrates. Our findings showed a higher FAE activity in the enzymatic extracts produced on corn cob (0.070 ± 0.004 U/mL). Nevertheless, the enzymatic extracts produced on VTS demonstrated a better performance for FA release from both corn cob (2.05 ± 0.01 mg/g) and VTS (0.19 ± 0.003 mg/g). This result was probably because of the higher xylanase/FAE ratio determined in VTS extract. Therefore, an additional assay was carried out by supplementing corn cob extract with a commercial xylanase to test the influence of FAE/xylanase ratio in FA release. The results revealed the relevance of the FAE/xylanase ratio for an optimal FA release.     

Growth and fat formation of Aspergillus oryzae and Aspergillus terreus on enzyme-hydrolyzed sweet potatoes

Two fungi, namely,Aspergillus oryzae andAspergillus terreus, have been grown on enzyme-hydrolyzed sweet potato medium either as such or supplemented with an external source of nitrogen in the form of ammonium carbonate, sodium nitrate, or asparagine. Hydrolysis alone promoted carbohydrate uptake, growth as well as fat formation to a remarkable extent in both fungi.Addition of an external supply of nitrogen to the hydrolyzed media increased further carbohydrate absorption which continued until the potato media were almost completely depleted; on unhydrolysed potato media, appreciable amounts of carbohydrates always remained unutilized. It also accelerated the rate of building up so that the dry mycelium increased by more than 4 times in some cases. Fat formation was consequently remarkably enhanced specially in the presence of ammonium carbonate or asparagine.     

Effect of pH and stirring rate on itaconate production by Aspergillus terreus

The production of itaconic acid from glucose-based media by Aspergillus terreus NRRL 1960 was found to be controlled by stirring rate and pH. When the phosphorous (P) level in the production medium was reduced to less than 10 mg l(-1), the fungal mycelium exhausted its primary growth and started to excrete itaconic acid, while it continued its secondary growth at the expense of ammoniacal nitrogen. The fermentation exhibited a mixed-growth-associated product formation kinetics, the non-growth associated production term (mI) being practically zero only when the pH was left free to change from 3.4 down to 1.85. On the contrary, when the pH was kept reducing up to a constant value by automatic addition of KOH 4 mol l(-1), the itaconate yield coefficient on the initial glucose supplied (Y(I/So)) and mI and were 0.53 g g(-1) and 0.028 h(-1) at pH 2.4 and 320 rev min(-1) and 0.5 g g(-1) and 0.036 h(-1) at pH 2.8 and 400 rev min(-1), respectively. Although the differences between mI and Y(I/So) were statistically insignificant at the 95% confidence level, the net difference in the corresponding yield coefficients for itaconic acid on mycelial biomass resulted in a maximum itaconate production rate of 0.41 g l(-1) h(-1) at pH 2.8 and 400 rev min(-1), thus showing that this operating condition is no doubt optimal for the process under study.     

One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.     

Biochemical studies on acetate non-utilizing mutants of Aspergillus terreus IRRL 16043

The strain Aspergillus terreus IRRL 16043 can utilize glucose as well as acetate as a sole carbon source. Thirty-nine mutants were isolated from the wild-type by treatment with a chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNTG) which could not utilize acetate as a sole carbon source, and were designated as acetate non-utilizing (acu). By complementation and biochemical analyses they were divided into three functional groups, acu A, acu B and acu C lacking isocitrate lyase, malate synthase and acetyl-CoA synthetase activity, respectively.     

Effect of Butyrolactone I on the Producing Fungus, Aspergillus terreus

Butyrolactone I [α-oxo-β-(p-hydroxyphenyl)-γ-(p-hydroxy-m-3,3-dimethylallyl-benzyl)-γ-methoxycarbonyl-γ-butyrolactone] is produced as a secondary metabolite by Aspergillus terreus. Because small butyrolactone-containing molecules act as self-regulating factors in some bacteria, the effects of butyrolactone I on the producing organism were studied; specifically, changes in morphology, sporulation, and secondary metabolism were studied. Threefold or greater increases in hyphal branching (with concomitant decreases in the average hyphal growth unit), submerged sporulation, and secondary metabolism were observed when butyrolactone I was added to cultures of A. terreus. Among the secondary metabolites whose production was increased by this treatment was the therapeutically important compound lovastatin. These findings indicate that butyrolactone I induces morphological and sporulation changes in A. terreus and enhances secondary metabolite production in a manner similar to that previously reported for filamentous bacteria.     

Effect of cultural factors on cellulase activity and protein production by Aspergillus terreus

Protein production by Aspergillus terreus GN1 grown on 1.0% alkali-treated bagasse was studied under various cultural conditions. The maximum biomass protein content of 20.1% and protein recovery of 11.2% was obtained with an initial pH of 4.0, with 1/5 (v/v) inoculum in continuously shaken cultures grown for seven days. Protein content of the alkali-treated bagasse was 3.0%. Highest crude protein percent also corresponded with highest carboxymethyl cellulase and filter paper enzyme activities.     

Molecular characterization and enzymatic hydrolysis of naringin extracted from kinnow peel waste

Kinnow peel, a waste rich in glycosylated phenolic substances, is the principal by-product of the citrus fruit processing industry and its disposal is becoming a major problem. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. In this work, infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from kinnow peel waste. Further, recombinant α-L-rhamnosidase purified from Escherichia coli cells using immobilized metal-chelate affinity chromatography (IMAC) was used for naringin hydrolysis. The purified enzyme was inhibited by Hg2+ (1 mM), 4-hydroxymercuribenzoate (0.1 mM) and cyanamide (0.1 mM). The purified enzyme established hydrolysis of naringin extracted from kinnow peel and thus endorses its industrial applicability for producing rhamnose.     

Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases

In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, beta-glucuronidase, non specific arylesterase, microsomal arylsulphatase, beta-galactosidase, beta-N-acetylglucosaminidase, acid alpha-glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.     

Immobilization of cellulase and d-xylanase complexes from Aspergillus terreus F-413 on controlled porosity glasses

The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g^?1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively.     

Diazonium inactivation in simultaneous-coupling and product inhibition in post-coupling azo-techniques for demonstrating activity of acid hydrolases

Summary In this communication results are presented of an investigation in which the activity of the hydrolytic enzymes acid phosphatase, -glucuronidase, non specific arylesterase, microsomal arylsulphatase, -galactosidase, -N-acetylglucosaminidase, acid -glucosidase and aminopeptidase M are demonstrated in tissue sections with simultaneous- and post-coupling azo-techniques. Semipermeable membrane techniques are used to hamper enzyme diffusion during the incubation period. From the histochemical and biochemical findings it appeared that an advantage of the post-coupling techniques over the simultaneous-coupling techniques is that inactivation of the enzymes by the coupling reagents is avoided. On the other hand post-coupling techniques are subject to product inhibition. With kinetic inhibition studies it is found that for microsomal arylsulphatase and non-specific arylesterase this product inhibition is non-competitive. This product inhibition may be a problem for histochemical quantitative post-coupling techniques for the determination of acid hydrolase activity.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes

Achievement of efficient enzymatic degradation of cellulose to glucose is one of the main prerequisites and one of the main challenges in the biological conversion of lignocellulosic biomass to liquid fuels and other valuable products. The specific inhibitory interferences by cellobiose and glucose on enzyme-catalyzed cellulose hydrolysis reactions impose significant limitations on the efficiency of lignocellulose conversion — especially at high-biomass dry matter conditions. To provide the base for selecting the optimal reactor conditions, this paper reviews the reaction kinetics, mechanisms, and significance of this product inhibition, notably the cellobiose and glucose inhibition, on enzymatic cellulose hydrolysis. Particular emphasis is put on the distinct complexity of cellulose as a substrate, the multi-enzymatic nature of the cellulolytic degradation, and the particular features of cellulase inhibition mechanisms and kinetics. The data show that new strategies that place the bioreactor design at the center stage are required to alleviate the product inhibition and in turn to enhance the efficiency of enzymatic cellulose hydrolysis. Accomplishment of the enzymatic hydrolysis at medium substrate concentration in separate hydrolysis reactors that allow continuous glucose removal is proposed to be the way forward for obtaining feasible enzymatic degradation in lignocellulose processing.     

Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by Aspergillus terreus

Summary Effect of aeration rate and impeller tip speed on mycelium growth and itaconic acid production was investigated in a batch culture of Aspergillus terreus IFO-6365. When impeller tip speed was 94.2 cm/sec at a fixed aeration rate of 0.5 vvm, itaconic acid concentration was 3.6 and 1.6 times higher than those in the impeller tip speed of 62.8 and 125.7 cm/sec, respectively. When an oxygen-enriched air was supplied at a fixed impeller tip speed of 94.2 cm/sec and dissolved oxygen concentration was maintained in the 20–60 % range, both itaconic acid concentration and mycelium growth were not affected by the dissolved oxygen concentration.