Purification of polygalacturonase from solid-state cultures of Aspergillus carbonarius

Purification of polygalacturonase (PG) from the cultures of Aspergillus carbonarius obtained by acetate buffer extraction after solid-state fermentation was attempted by integrated membrane process and alginate affinity precipitation. The carbohydrates were completely eliminated (98%–99%) with a PG recovery of 72%–80% during integrated membrane process, which would otherwise interfere with the purification process and lead to enzyme loss. However, specific activity of PG did not improve (1.19–1.21 fold) due to the presence of other similar molecular mass proteins. Under optimum conditions of affinity precipitation, the specific activity of PG enhanced to 2450 U/mg (4 fold) with almost complete elimination of carbohydrates and colour compounds resulting in a PG recovery of 61%. PG purity obtained with ultrafiltration (UF) was comparable with the conventional dialysis during desalting eluted PG, besides UF rendered a concentrated PG. The enzyme purity stated was as descend by SDS–PAGE. The results suggested suitability of affinity precipitation for PG purification from solid-state cultures and the potential of UF as a single step process for handling eluted PG.     

Purification and characterisation of Aspergillus sojae naringinase: The production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG-CoA reductase

Aspergillus sojae isolated from a traditional Korean fermented soybean product exhibited strong naringinase activity. The naringinase enzyme was purified and had a molecular weight of 70 kDa. The α-l-rhamnosidase activity of this enzyme was optimal at pH 6.0 and stable in the pH range of 5.5–8.0. The purified enzyme also had β-d-glucosidase activity, but the activity was relatively weak compared to the activity of the naringinase from Penicilliumdecumbens. The enzymatic bioconversion by A. sojae naringinase of naringin to prunin was efficiently performed with a 91% yield and a negligible amount of naringenin. The bioconversion was achieved by repetitive batch reactions with enzyme recycling. Prunin exhibited a markedly enhanced solubility compared to naringenin and naringin while maintaining the in vitro inhibition of HMG-CoA reductase. The results reported in this paper show that the naringinase produced by A. sojae will be useful in enhancing the potential bioavailability of naringin by efficiently converting it to prunin as a food component in citrus.     

Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves

We report purification and characterisation of a polyphenol oxidase from red Swiss chard (rcPPO). Our purification procedure resulted in a 39-fold enrichment in specific activity and 17% recovery of total enzyme activity. The purified rcPPO appeared as a monomeric protein of 41 kDa, with a specific conformation conserved in the Cu²⁺ combining region. It was optimally active at pH 7.5 and 45 °C. It had a diphenolase substrate preference towards l-DOPA, catechol and chlorogenic acid, but also exhibited weak monophenolase one toward 4-methoxyphenol and l-tyrosine. We also found that the enzyme was activated by K⁺, Na⁺, SDS and laurouyl sarcosine, but inhibited by divalent cations including Ca²⁺, Cu²⁺. Its activity was completely inhibited by ascorbic acid, cysteine, 1,4-dithiothreitol, β-mercaptoethanol, sodium diethyldithiocarbamate, sodium metabisulphite, sodium sulphite and thiourea. This first report on the purification and characterisation of red Swiss chard PPO provides a basis for understanding and use of this enzyme.     

Physicochemical characterisation of β-chitosan from Sepioteuthis lessoniana gladius

β-Chitin and its chitosan from the gladius of Sepioteuthis lessoniana have been isolated, purified, characterised and compared with the commercial chitosan. Ash, moisture, mineral, metal and elemental content were analyzed using standard techniques. The optical activity of chitin was found to be levorotatory. The degree of deacetylation was calculated by potentiometric titration and ¹H NMR. Viscosity average molecular weight of β-chitosan was calculated by viscometry and size average molecular weight by GPC. The structure of β-chitosan was elucidated with FT-IR and NMR. Thermal nature, crystalline structure and morphology of β-chitosan were characterised through DSC, XRD and SEM, respectively. The water and fat binding capacity of β-chitosan presently studied was significantly higher than that of the commercial chitosan. The result of the present study adds that S. lessoniana gladius is also an additional source of β-chitin and chitosan of higher yield, lower molecular weight and higher degree of deacetylation.     

Effect of Capsicum carotenoids on growth and aflatoxins production by Aspergillus flavus isolated from paprika and chilli

The aim of this study was to determine the effect of a carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and aflatoxins (AF) production of AF-producing Aspergillus flavus isolates.     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Partial purification and characterisation of endoglucanase from an edible mushroom, Lepista flaccida

A crude extract was prepared from the fruiting body of Lepista flaccida, an edible mushroom and endoglucanase activity of the extract was increased 14-fold with ammonium sulphate precipitation. Maximum enzyme activity was seen at pH 4.0 and 50 °C when carboxymethylcellulose was used as a substrate. K_0.5 and V_max values of the partially purified endoglucanase were 7.7 mg/ml and 25 ± 0.9 U/mg protein, respectively. The enzyme was quite stable over a broad range of pH (2.0–9.0) at 4 °C. When it was incubated at temperatures between 20 °C and 60 °C for 12 h, it conserved much of its original activity (over 40%). The activity of the enzyme increased by 234 ± 3.6% in the presence of 1 mM Mn²⁺. The endoglucanase was inhibited by EDTA, PMSF, β-ME and DDT. In conclusion, pH and thermal stability of the L. flaccida endoglucanase could make it useful for industrial purposes.     

Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.)

Lipoxygenase (LOX) from pea seeds (Pisum sativum var. Telephone L.) was extracted and studied of biochemical properties. The molecular mass of purified lipoxygenase was 93 kDa. The effects of substrate specificity, pH, and sensibility to various inhibitors: caffeic acid, ferulic acid, benzoic acid, catechin, quercetin and kaempferol of LOX were investigated. Lipoxygenase showed the highest activity toward linoleic acid and the lowest toward oleic acid as substrates. Kinetic studies indicated that V_max of the LOX activity was 151.5 U/min and corresponding K_m value of 0.44 × 10⁻³ M. Optimum pH of lipoxygenase was reported at 5.5. Caffeic acid was the most effective inhibitor and kaempferol was the least effective.     

Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes

Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. Besides, OTA production is a very consistent property of this species and for this reason atoxigenic isolates of A. carbonarius are very rarely found in natural environments. In the present study, for the first time, three nonochratoxigenic wild strains of A. carbonarius have been discovered, unambiguously identified, characterized in deep and compared to ochratoxigenic strains of the same species. In addition, polyketide synthase (pks) genes suggested to be involved in OTA biosynthesis were also screened in these strains. The identification of the strains was confirmed by ITS-5.8S rRNA, β-tubulin and calmodulin gene sequencing. The three atoxigenic strains did not produce OTA in a conducive culture medium at any of the temperatures and times of incubation tested. Five ketosynthase domains from pks genes previously described in A. carbonarius were detected both in ochratoxigenic and in nonochratoxigenic strains. Atoxigenic strains of A. carbonarius could be useful as biotechnological agents to be used in food industry and as biological agents for control of OTA production in vineyards and other crops.     

Cloning of Aspergillus oryzae Aovps5 gene,homologous to vacuolar protein sorting associated gene VPS5 and construction of the disruptant

Aovps5 gene was isolated from Aspergillus oryzae as a homologue to S. cerevisiae VPS5 gene which encodes a polypeptide consisting of 451 amino acids that is nearly 32% homologous to Vps5p. Three Aovps5 gene disruptants were generated and they showed higher activity of tripeptidyl peptidase, which is mainly detected in vacuoles, in their culture medium. Higher amount of nitrogenous constituent was found in the filtrate of wheat gluten degraded by addition of culture medium of these disruptants than that of wild type strain. These results suggest that disruption of Aovps5 may contribute to production of fermented foods.     

Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341

Aspergillus oryzae (MTCC 5341) has the largest expanse of hydrolytic genes, that includes 135 protease genes coding for alkaline, acid as well as neutral proteases. This study reports the purification and characterisation of an acid protease obtained from A. oryzae MTCC 5341. A. oryzae MTCC 5341 produces one of the highest reported acid protease activities reported so far (8.3 × 10⁵ U/g dry bran). The extracellular acid protease (47 kDa) was found to be active in the pH range 3.0–4.0 and stable in the pH range 2.5–6.5. Optimum temperature for activity was 55 °C. The protease was purified 17–fold with a yield of 29%. The enzyme was characterised to be an aspartate protease by inhibition studies, using pepstatin and its ability to activate trypsinogen. The enzyme cleaved the B-chain of insulin at L–V and Y–T residues.     

A heat-stable trypsin from the hepatopancreas of the cuttlefish (Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins.     

Purification and characterisation of two enzymes related to endogenous formaldehyde in Lentinula edodes

In this study, γ-glutamyl transpeptidase (GGT) and l-cysteine sulphoxide lyase (C-S lyase) were purified from the fruiting body of Lentinula edodes in three steps and then characterised. We found that GGT together with C-S lyase caused the generation of endogenous formaldehyde in L. edodes. GGT was composed of a large subunit of 41 kDa and a small subunit of 25 kDa, and C-S lyase was composed of two identical subunits of 46 kDa, as determined by SDS–PAGE. GGT was stable at pH 8.0–10.0 with an optimum pH of 8.8, and was stable at 20–50 °C with an optimum activity at 37 °C. C-S lyase was stable at pH 8.0–9.0 with an optimum pH of 8.5, and was stable at 20–60 °C with an optimum activity at 40 °C. The present work supports the study of the mechanism of endogenous formaldehyde in L. edodes.     

A novel aspartic protease from the viscera of Sardinelle (Sardinella aurita): Purification and characterisation

A novel aspartic protease was extracted from the defatted viscera of sardinelle (Sardinella aurita) and purified, with a 9.5-fold increase in specific activity and 23.3% recovery. The molecular weight of the purified enzyme was estimated to be 17 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The purified enzyme appeared as a single band on native-PAGE. The optimum pH and temperature for protease activity were around 3.0 and 40 °C, respectively. The enzyme showed pH stability between 2.0 and 5.0 and retained more than 50% of its activity after heating for 30 min at 50 °C. The enzyme lost 90% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride. Its K_m value was determined to be 0.73 × 10⁻⁴ M using haemoglobin as a substrate. The N-terminal 12 amino acid sequence of the purified acidic protease was R V I I E D X D Q F C T. This sequence showed low homology with aspartic peptidases of several other species of fish, suggesting that the enzyme is a new aspartic protease.     

Isolation and characterisation of collagens from the skin of largefin longbarbel catfish (Mystus macropterus)

Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from the skin of largefin longbarbel catfish (Mystus macropterus) with yields of 16.8% and 28.0%, respectively, on the basis of dry weight. Both ASC and PSC contained α1 and α2 chains and the amino acid composition of collagen was close to that of calf skin type ? collagen. The intrinsic viscosities of ASC and PSC were 14.9 dl/g and 14.5 dl/g, respectively. Similar ultraviolet and FTIR spectra of ASC and PSC were observed. However, peptide maps of ASC and PSC, hydrolysed by trypsin, revealed some differences in primary structures between the two fractions. Denaturation temperatures of ASC and PSC were 32.1 °C and 31.6 °C, respectively. The higher T_m showed that it is possible to use largefin longbarbel catfish skin collagen as an alternative source of vertebrate collagens for industrial purposes.     

Optimisation of antioxidants extraction from soybeans fermented by Aspergillus oryzae

The extraction of antioxidant compounds from soybeans fermented with Aspergillusoryzae was optimised using a factorial design. A kinetic study of the total phenolic production and DPPH radical scavenging activity was first performed at the points selected in the factorial design. In both cases, the experimental profiles were fitted to a modified first-order kinetic model. To investigate the combined effects of temperature and solvent concentration on the extraction, the parameters obtained from the fitted kinetic models were used as response variables in a rotatable second-order design with quintuple replications in the centre of the experimental domain. The results obtained indicate that temperature had the most significant effect. The response surfaces show a maximum in the experimental domain studied. The optimum conditions for the extraction of total phenolic content were 65.3 °C and 73.1% ethanol, in which 56.2 mg of GAE/g were predicted. A scavenging activity of 81.6% DPPH radical was predicted at the optimum conditions of 61.6 °C and 60% ethanol.