The understanding that enzymatic degradation of fruit pectin can clarify juices and improve juice yields resulted in the search for microbial pectinases and application in vegetable- and fruit-processing industries. Identified enzymes were classified on the basis of their catalytic activity to pectin or its derivatives and in terms of industrial use. Discovery of gene sequences that coded the enzymes, protein engineering, and molecular biology tools resulted in defined microbial strains that over-produced the enzymes for cost-effective technologies. Recent perspectives on the use of pectin and its derivatives as dietary fibers suggest enzymatic synthesis of the right oligomers from pectin for use in human nutrition. While summarizing the activities of pectin-degrading enzymes, their industrial applications, and gene sources, this review projects another application for pectinases, which is the use of enzymatically derived pectin moieties in functional food preparation.

Integrated membrane process (IMP) and alginate affinity precipitation (AAP) were assessed for the purification of Aspergillus carbonarius polygalacturonase (PG) obtained after submerged (SmF) and solid-state fermentations (SSF). IMP enhanced the purity (4.69 fold) and recovery (76%) of SmF-PG, but did not improve the purity of SSF and commercial PG due to the presence of other similar molecular mass proteins. AAP enhanced the PG purity and recovery in all the PG samples (SmF, SSF and commercial PG). IMP offers several process advantages over AAP but not suitable for purification of all types of PGs. Our studies on SmF- and SSF-PG had shown that downstream process needs are specific for individual cases and warrants careful considerations.

Microfiltration (MF) and ultrafiltration (UF) membranes were screened for improving the specific activity of polygalacturonases (PG) in the culture broth of Aspergillus carbonarius obtained after submerged fermentation. While 200 and 450 nm MF membranes eliminated some of the larger non-enzymatic proteins, 50 kDa UF membrane exhibited a marginal selectivity between the enzyme and other smaller proteins. The 450 nm MF and 50 kDa UF membranes selected were further evaluated under different process conditions for an integrated membrane process. The process efficacy of three different schemes was also studied for enzyme purification. A two-stage membrane process employing MF followed by UF improved the enzyme-specific activity (5,590 U/mg) by 4.69-fold eliminating the larger and smaller non-enzymatic proteins as well as non-protein impurities with a recovery of 76% enzymes, besides resulting in higher productivity. Thus, adoption of integrated membrane process with appropriate selection of membranes could result in high recovery of enzymes with improved specific activity.

BACKGROUND: Microfiltration (MF: 70-450 nm) and ultrafiltration (UF: 10-500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes.RESULTS: MF resulted in removal of 2-25% carbohydrates with an enzyme recovery of 69-82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg(-1) of dry matter.CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. (C) 2008 Society of Chemical Industry.

The methanolic extract of Monascus purpureus cultivated by solid-state fermentation on rice showed strong 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and better yield as compared to other polarity based extracted fractions. It was selected for further purification of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on the spectroscopic analysis by UV, IR, 1H NMR, 13C NMR, 2D-HSQCT NMR, and MS, the antioxidant isolated was elucidated as a derivative of dihydromonacolin-K, where the ester group is 2-methyl propionate, designated as dihydromonacolin-MV. The DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC50 20±1 μg ml−1). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a liposome model with an IC50 value of 5.71±0.38 μg ml−1 and superoxide radical scavenging activity with an IC50 value of 163.97±2.68 μg ml−1.

Aspergillus carbonarius, an ascomycetes fungus, is known to produce pectinase in solid-state fermentation. A mutant strain of A. carbonarius UV-10046 selected for temperature tolerance over produced polygalacturonase and during growth accumulated an yellow pigment in its biomass. Since the colored fungus suggested its application for food use, the freeze-dried biomass was evaluated to assess its safety in experimental animals. Acute and sub-acute toxicity studies were conducted on both sexes of albino rats. Feeding acute doses of A. carbonarius freeze-dried biomass at 0.5–5.0 g/kg body weight to adult rats did not show any symptoms of toxicity or mortality of the rats. Similarly, dietary feeding of A. carbonarius at 0.25–2.0% level (w/w) for 14 weeks did not produce any significant changes in food intake or gain in body weight of the experimental rats compared to control rats. There were no significant differences in the relative weight of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum enzyme levels between the experimental and control groups. The results clearly showed that acute and sub-acute oral feeding of freeze-dried whole cells of A. carbonarius mutant for 14 weeks did not produce any toxic effects in male and female rats.

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent α-glucosidase inhibitor. The enzyme production was induced when A. niger was grown in starch medium containing the inhibitor. Antibodies against the precursor enzyme cross-reacted with the 54-kDa Taka-amylase protein of A. oryzae. It resembled Taka-amylase in most of its properties and also hydrolyzed starch to maltose of α-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Aims: Characterization of dehydromonacolin-MV2, a bioactive metabolite isolated from Monascus purpureus mutant (CFR 410-11).Methods and Results: Chloroform extract of rice, fermented with a hyperpigment-producing mutant of M. purpureus (CFR 410-11) was found to contain metabolites that inhibited the growth of Bacillus, Pseudomonas and Streptococcus in agar gel diffusion assays. The extract inhibited lipid peroxidation and scavenged 2,2-diphenyl-1-pycrylhydrazyl and hydroxyl radicals. The active compound purified by silica gel column chromatography was characterized by NMR. The carbon, proton and 2D HSQCT assignments identified dehydromonacolin-MV2 as the bioactive metabolite.Conclusions: Dehydromonacolin-MV2 apparently originated in the mutant by hydroxylation and oxidation of monacolin-J, an intermediate of monacolin biosynthetic pathway.Significance and the Impact of the Study: Identification of the production of dehydromonacolin-MV2 by M. purpureus mutant (CFR 410-11) is new to literature. Bioactive properties of the compound suggested its pharmaceutical applications.

Raw materials of plant origin are processed in a variety of ways for the production of food. Modern food processing uses enzyme preparations to improve pulp and juice yields from fruits, in wine preparation to increase the color and quality of the wine, in tea manufacture for flavor release, in oil extraction from olives, and in steeping maize for the recovery of sugar. In each of these processes, pectin degradation is an important step. Pectin is a complex colloidal carbohydrate found in plants. Pectin is found in the middle lamella and primary cell walls of higher plants and is made of anhydrogalacturonic acid units. The carboxyl groups of galacturonic acid in pectin may be partly esterified by methyl groups or partly or totally neutralized by one or more bases. Pectic substances are produced in plants from a water insoluble parent pectic substance called

The observation that a mutant strain of Aspergillus niger isolated for protease overproduction accumulated Taka-amylase supported an earlier report that processing of the precursor amylase by protease resulted in the secretion of multiple amylases. Studies using a mutant strain revealed that such processing was not due to aspergillopepsin but to autocatalysis by an inherent protease activity of the precursor and glucoamylase. Alignment of protease sequences with glucoamylase showed regions of consensus with serine carboxypeptidase of A. niger. Thus point mutations in this region due to ultraviolet radiation apparently caused the mutant to evolve with enhanced protease activity that degraded the precursor and accumulated Taka-amylase.

A strain of Bacillus produced an amylase with properties characteristically different from known bacterial amylases. The purified 80 kDa protein of pI 5.1 dextrinized starch, glycogen and pullulan. The temperature and pH optima of the enzyme were 60 °C and 6.6 respectively. In the presence of 0.05 M CaCl 2 , the enzyme retained stability for 15 min at 80 °C. Antibodies raised to the amylase protein showed no reaction with α -amylases of Bacillus sp. and B. licheniformis . In culture, proteolytic degradation of the enzyme was observed.

Low pectinase production by Aspergillus carbonarius growing on wheat bran solid substrate was found to be due to reduced colonizational ability of the fungus. Since A. niger showed higher growth rates on wheat bran, strain improvement to obtain higher pectinase production in solid state was carried out by inter-specific fusion of protoplasts of A. carbonarius and A. niger. One of the mutants selected for higher activities of alpha-glucosidase showed improved growth rates on wheat bran solid substrate together with increased pectinase production. Size similarities of amplified polymorphic DNA of the mutant with the two parents and identification of a 66 kDa polygalacturonase specific to A. niger suggested genetic recombination in the mutant.

A 125‐kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673–675] was also found to possess activity towards raw starch. Segregation of these activities in the 71‐kDa glucoamylase and a 53‐kDa α‐amylase‐like enzyme supported by antibody cross‐reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125‐kDa starch hydrolysing enzyme as their precursor. N‐terminal sequence analysis further revealed that the 71‐kDa glucoamylase was the N‐terminal product of the precursor enzyme. Immunological cross reactivity of the 53‐kDa amylase with antibodies raised against the precursor enzyme but not with the 71‐ and 61‐kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C‐terminal fragment of the precursor. The N‐terminal sequence of the 53‐kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross‐reactivity to a 10‐kDa non‐enzymic peptide and a 61‐kDa glucoamylase described these proteins as products of the 71‐kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.

A major isoform of β-1,3-glucanase from pearl millet seedlings was purified following ammonium sulfate precipitation, ion-exchange chromatography and gel filtration techniques. The enzyme had a molecular weight of 20.5 kDa on SDS–PAGE and was highly basic with a pI of 9.6. It was thermostable with a broad temperature optima for activity ranging from 37 to 70°C and had an optimum pH of 5.2. Mercuric chloride and para-chloromercuric benzoate inhibited completely the enzyme while manganese chloride activated it. Antibodies raised against the purified β-1,3-glucanase identified another protein with an apparent molecular weight of 30 kDa in western reactions. Significance of this enzyme in pearl millet–downy mildew host–pathogen interaction is discussed.

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 degrees C and glucose, maltose and maltodextrins at 70 degrees C as primary products, suggested significant applications for the enzyme in starch-processing industries.

A procedure to detect raw and gelatinized starch activities of glucoamylase on isoelectric focusing (IEF) gels by using 2, 3, 5‐triphenyltetrazolium chloride is described. The reagent reacts with the reducing group of glucose released by glucoamylase from the substrate starch. Using the reaction, production of glucoamylase by Aspergillus niger was detected on 10% IEF gels within a pH range of 2.5—9.5. Since the method can detect raw and gelatinized starch activities of glucomylase associated with 1 μg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.

An enzyme-linked immunosorbent assay for sensitive, specific and quantitative estimation of fungal biomass during solid-state fermentation is described. Using this method, differential growth rates and colonization of the substrate can be studied. The assay has potential application for the efficient monitoring of solid-state fermentation involving specific fungus, for which available methods are not adequate.

A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673–675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa α-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.

MIG1, encoding a C2H2 zinc-finger repressor protein involved in carbon catabolite repression, was found to play a role in non-sexual flocculation of Saccharomyces cerevisiae. Disruption of MIG1 in a flocculent mutant strain of NCYC 227, resulted in a non-flocculent phenotype. Expression of MIG1 on a 2 mu pRS426 vector in a non-flocculent strain, YM 4134, caused flocculation; MIG1 on a high-copy-number LEU2-d plasmid caused intense flocculation in the same strain. Mutations in the SSN6 and TUP1 genes confer a flocculent phenotype in non-flocculent strains of S. cerevisiae, and it has been shown that Mig1 can tether the Ssn6p-Tup1p complex to the regulatory regions of glucose-repressible genes. Mutations in tup1 in a MIG1 background caused flocculation while double mutants of TUP1 and MIG1 did not flocculate. Based on these results, a model for the role of MIG1 in flocculation gene regulation is proposed.