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Biomedical subjects

C A Reddy

Publications and source records attributed to C A Reddy.

At least 55 records · Page 3Linked to original sources
PubMed1990

Characterization of a new lignin peroxidase gene (GLG6) from Phanerochaete chrysosporium.

Sequence analysis of a new lignin peroxidase (LIP) gene, GLG6, from P. chrysosporium showed that it encodes a mature LIP protein with a predicted Mr of 36,454. A 28 aa signal peptide precedes the mature protein. The coding region of GLG6 is interrupted by nine introns ranging in size from 50-57 bp. GLG6 encoded-LIP has 72%, 88% and 82% homology, respectively, to the LIP isozymes H2, H3, and H10. Comparison of the N-terminal sequence of GLG6-encoded LIP to that of the LIP proteins H2, H8, H10 also showed that the former is relatively less related to the H2 protein than it is to the H8 and H10 proteins. Expression of GLG6, similar to the other LIP genes, occurs only during secondary metabolism.

Amino Acid Sequence↗
PubMed1990

Characterization of extracellular peroxidases produced by acetate-buffered cultures of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Growth of Phanerochaete chrysosporium in a nitrogen-limited medium buffered with sodium acetate, instead of the commonly used 2,2-dimethylsuccinate (DMS), resulted in quantitative and qualitative differences in the production of various extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) involved in lignin degradation. The results indicate that production of LIPs and MNPs can be selectively enhanced by manipulation of culture conditions. Partial N-terminal analyses of the major LIPs and MNPs have made it possible to assign a specific protein to the specific genes and cDNAs that have been reported recently. The LIPs and MNPs differed widely in their ability to decolorize various dyes that are known to be degraded by the lignin degrading enzyme system of P. chrysosporium.

Acetates↗
PubMed1990

Nitrogen-deregulated mutants of Phanerochaete chrysosporium--a lignin-degrading basidiomycete.

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (14C-synthetic lignin----14CO2), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the other hand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H2O2 in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗
PubMed1990

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.

Basidiomycota↗
PubMed1990

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium.

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.

Basidiomycota↗
PubMed1989

Use of a shuttle vector for the transformation of the white rot basidiomycete, Phanerochaete chrysosporium.

A novel shuttle vector based spheroplast transformation system for the lignin degrading filamentous fungus P. chrysosporium is described. The transformation vector, designated pRR12, consists of the yeast integration plasmid YIp5, a putative autonomous replication sequence (ars) of P. chrysosporium, and a 2.2 kb PvuII fragment carrying kanr determinant from plasmid pNG35, which confers resistance against both kanamycin and the related antibiotic G418. Two different strains of P. chrysosporium (ME446 and BKM-F) were transformed to G418 resistance using vector pRR12. Approximately 20 transformants per micrograms of vector DNA were obtained. The transforming vector pRR12 could be recovered from the total DNA of transformants by E. coli transformation, albeit at a low frequency.

Basidiomycota↗
PubMed1987

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium.

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.

Amino Acid Sequence↗
PubMed1986

Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes.

Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E. coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides. Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.

Amino Acid Sequence↗
PubMed1986

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae.

DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or PstI. This cloned fragment hybridized with the total DNA from C. utilis and from Leu+ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI-HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.

Candida↗
PubMed1986

Optimized batch fermentation of cheese whey-supplemented feedlot waste filtrate to produce a nitrogen-rich feed supplement for ruminants.

An optimized batch fermentation process for the conversion of cattle feedlot waste filtrate, supplemented with cheese whey, into a nitrogenous feed supplement for ruminants is described. Feedlot waste filtrate supplemented with cheese whey (5 g of whey per 100 ml) was fermented by the indigenous microbial flora in the feedlot waste filtrate. Ammonium hydroxide was added to the fermentation not only to maintain a constant pH but also to produce ammonium salts of organic acids, which have been shown to be valuable as nitrogenous feed supplements for ruminants. The utilization of substrate carbohydrate at pH 7.0 and 43 degrees C was >94% within 8 h, and the crude protein (total N x 6.25) content of the product was 70 to 78% (dry weight basis). About 66 to 69% of the crude protein was in the form of ammonia nitrogen. Lactate and acetate were the predominant acids during the first 6 to 8 h of fermentation, but after 24 h, appreciable levels of propionate and butyrate were also present. The rate of fermentation and the crude protein content of the product were optimal at pH 7.0 and decreased at a lower pH. For example, fermentation did not go to completion even after 24 h at pH 4.5. Fermentation proceeded optimally at 43 degrees C, less so at 37 degrees C, and considerably more slowly at 23 and 50 degrees C. Concentrations of up to 15 g of cheese whey per 100 ml of feedlot waste filtrate were fermented efficiently. Fermentation of feedlot waste filtrate obtained from animals fed low silage-high grain, high silage-low grain, or dairy rations resulted in similar products in terms of total nitrogen and organic acid composition.

Journal Article↗
PubMed1986

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.

Aspergillus niger↗
PubMed1986

Purification and characterization of glucose oxidase from ligninolytic cultures of Phanerochaete chrysosporium.

Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).

Basidiomycota↗
PubMed1985

Lack of lignin degradation by glucose oxidase-negative mutants of Phanerochaete chrysosporium.

Glucose oxidase-negative (gox-) mutants of Phanerochaete chrysosporium were isolated after exposing conidia to UV irradiation. The gox- mutants exhibited little or no ability to degrade lignin (2-[14C]-synthetic lignin to 14CO2); however, they retained other secondary metabolic features such as the ability to conidiate and produce veratryl alcohol, suggesting that they are not pleiotropic for secondary metabolism. Lignin degradation activity was restored in gox+ revertants. These results, in support of earlier evidence, indicate that glucose oxidase activity plays an important role in lignin degradation by P. chrysosporium.

Basidiomycota↗
PubMed1984

DNA sequences from a ligninolytic filamentous fungus Phanerochaete chrysosporium capable of autonomous replication in yeast.

Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated. These hybrid plasmids with the autonomous replication sequences from P. chrysosporium are maintained extra-chromosomally, are mitotically unstable and transform Ura3 deletion mutant of S. cerevisiae to Ura+ phenotype with high frequency. The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P. chrysosporium genomic DNA. Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning.

Base Sequence↗