Improving specific activity of Aspergillus carbonarius polygalacturonase using polymeric membranes.

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.

Characterization of dehydromonacolin-MV2 from Monascus purpureus mutant.

AIM: 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 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.

Safety evaluation of pigment containing Aspergillus carbonarius biomass in albino rats.

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 0.5-5.0g/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 proteolytic product of precursor starch-hydrolyzing enzyme of Aspergillus niger has Taka-amylase-like activity.

The 53-kDa amylase secreted by Aspergillus niger due to proteolytic processing of the precursor starch-hydrolyzing enzyme was resistant to acarbose, a potent alpha-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 alpha-anomeric configuration. However, it did not degrade maltotriose formed during the reaction and was not inhibited by zinc ions.

Evidence that cleavage of the precursor enzyme by autocatalysis caused secretion of multiple amylases by Aspergillus niger.

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.

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol.

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.

Evidence that the glucoamylases and alpha-amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme.

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 alpha-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.

Genetic improvement of Aspergillus carbonarius for pectinase overproduction during solid state growth.

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.

Separation and direct detection of raw and gelatinized starch hydrolyzing activities of glucoamylase on isoelectric focusing gels.

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 microg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.

MIG1 overexpression causes flocculation in Saccharomyces cerevisiae.

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.

Mitochondrial NADH dehydrogenase activity and ability to tolerate acetaldehyde determine faster ethanol production in Saccharomyces cerevisiae.

In rapidly fermenting yeast, the rotenone insensitive mitochondrial NADH dehydrogenase was not completely repressed by high glucose. This activity appeared to enhance the glycolytic rate due to which acetaldehyde accumulated intracellularly. To overcome the toxicity of acetaldehyde, the strain produced stress proteins. During late stationary phase of growth, the accumulated acetaldehyde was converted to ethanol resulting in faster ethanol production.

A surface lectin associated with flocculation in brewing strains of Saccharomyces cerevisiae.

A cell-wall-surface protein purified from the cells of Saccharomyces cerevisiae NCYC 227 was found to be involved in the non-sexual flocculation of this yeast. This 13 kDa protein was found to bind specifically to mannose. The protein bound to mannans isolated from yeast as well as in situ to intact cells, but only in the presence of calcium ions. The protein, a mannoprotein, formed aggregates as revealed in SDS-PAGE. Urea and higher temperatures prevented protein aggregation, suggesting that the flocculation of S. cerevisiae is primarily due to hydrogen bonding between mannan and protein.

Cell wall antigenic homology in certain species of Saccharomyces and an enzyme-linked immunosorbent assay for rapid identification of S. cerevisiae.

Cell-wall antigenic studies on Saccharomyces species grown by fermentation in 20% glucose broth indicated S. cerevisiae to develop two antigens specific for this species. These antigens were heat-stable polysaccharides and agglutination studies using specific antiserum prepared by cross absorption indicated their localization on the cell-wall surfaces. Using species-specific antibody, the results presented describe a specific and rapid identification of S. cerevisiae by ELISA according to the classification system of Yarrow (1984).

The effect of ethanol on cell wall antigens of Saccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast, making use of a serological technique.

Generally, natural isolates of high ethanol producing Saccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against whole Saccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation of S. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grown S. cerevisiae indicated the occurrence of thermolabile antigens common to Saccharomyces species. Higher concentrations of these antigens could be detected in those S. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producing S. cerevisiae could be identified.