Characterization of the novel antifungal chitosanase PgChP and the encoding gene from Penicillium chrysogenum

The protein PgChP is a new chitosanase produced by Penicillium chrysogenum AS51D that showed antifungal activity against toxigenic molds. Two isoforms were found by SDS-PAGE in the purified extract of PgChP. After enzymatic deglycosylation, only the smaller isoform was observed by SDS-PAGE. Identical amino acid sequences were obtained from the two isoforms. Analysis of the molecular mass by electrospray ionization-mass spectrometry revealed six major peaks from 30 to 31 kDa that are related to different levels of glycosylation. The pgchp gene has 1,146 bp including four introns and an open reading frame encoding a protein of 304 amino acids. The translated open reading frame has a predicted mass of 32 kDa, with the first 21 amino acids comprising a signal peptide. Two N glycosylation consensus sequences are present in the protein sequence. The deduced sequence showed high identity with fungal chitosanases. A high level of catalytic activity on chitosan was observed. PgChP is the first chitosanase described from P. chrysogenum. Given that enzymes produced by this mold species are granted generally recognized as safe status, PgChP could be used as a food preservative against toxigenic molds and to obtain chitosan oligomers for food additives and nutraceuticals.     

The glycolytic pathway of <Emphasis Type="Italic">Trimastix pyriformis</Emphasis> is an evolutionary mosaic

Background  

Glycolysis and subsequent fermentation is the main energy source for many anaerobic organisms. The glycolytic pathway consists of ten enzymatic steps which appear to be universal amongst eukaryotes. However, it has been shown that the origins of these enzymes in specific eukaryote lineages can differ, and sometimes involve lateral gene transfer events. We have conducted an expressed sequence tag (EST) survey of the anaerobic flagellate Trimastix pyriformis to investigate the nature of the evolutionary origins of the glycolytic enzymes in this relatively unstudied organism.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

Isolation,characterization and heterologous expression of a novel chitosanase from <Emphasis Type="Italic">Janthinobacterium</Emphasis> sp. strain 4239

Background  

Chitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.     

Transcription factors Elk-1 and SRF are engaged in IL1-dependent regulation of <Emphasis Type="Italic">ZC3H12A</Emphasis> expression

Background  

MCPIP is a novel CCCH zinc finger protein described as an RNase engaged in the regulation of immune responses. The regulation of expression of the gene coding for MCPIP - ZC3H12A is poorly explored.     

<Emphasis Type="Italic">Starmerella bombicola</Emphasis> influences the metabolism of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

Background  

The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.     

The tricarboxylic acid cycle in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> is independent of Fur and RyhB control

Background  

It is well established in E. coli and Vibrio cholerae that strains harboring mutations in the ferric uptake regulator gene (fur) are unable to utilize tricarboxylic acid (TCA) compounds, due to the down-regulation of key TCA cycle enzymes, such as AcnA and SdhABCD. This down-regulation is mediated by a Fur-regulated small regulatory RNA named RyhB. It is unclear in the γ-proteobacterium S. oneidensis whether TCA is also regulated by Fur and RyhB.     

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNA<Superscript>Asn</Superscript>

Background  

Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression.     

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in <Emphasis Type="Italic">E. coli</Emphasis>

Background  

Biotin is an essential enzyme cofactor that acts as a CO₂ carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for ³⁵S labeling of biotin.     

LmxMPK4, a mitogen-activated protein (MAP) kinase homologue essential for promastigotes and amastigotes of <Emphasis Type="Italic">Leishmania mexicana</Emphasis>

Background  

Leishmania parasites undergo profound morphological and biochemical changes while passing through their life cycle. Protein kinases have been shown to be involved in the differentiation from the extracellular flagellated promastigotes to the intracellular "non-flagellated" amastigotes and vice versa. Moreover, these enzymes are likely involved in the regulation of the proliferation of the different life stages.     

Expression profiling and integrative analysis of the <Emphasis Type="Italic">CESA</Emphasis>/<Emphasis Type="Italic">CSL</Emphasis> superfamily in rice

Background  

The cellulose synthase and cellulose synthase-like gene superfamily (CESA/CSL) is proposed to encode enzymes for cellulose and non-cellulosic matrix polysaccharide synthesis in plants. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, the global expression profiling patterns and functions of the OsCESA/CSL superfamily remain largely unknown.     

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

Background  

Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments.     

Evolution of plant RNA polymerase IV/V genes: evidence of subneofunctionalization of duplicated <Emphasis Type="Italic">NRPD2/NRPE2</Emphasis>-like paralogs in <Emphasis Type="Italic">Viola</Emphasis> (Violaceae)

Background  

DNA-dependent RNA polymerase IV and V (Pol IV and V) are multi-subunit enzymes occurring in plants. The origin of Pol V, specific to angiosperms, from Pol IV, which is present in all land plants, is linked to the duplication of the gene encoding the largest subunit and the subsequent subneofunctionalization of the two paralogs (NRPD1 and NRPE1). Additional duplication of the second-largest subunit, NRPD2/NRPE2, has happened independently in at least some eudicot lineages, but its paralogs are often subject to concerted evolution and gene death and little is known about their evolution nor their affinity with Pol IV and Pol V.     

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene,bioinformatics and biochemical characterization of the recombinant enzyme

Aims

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Methods and Results

A 1539 complete open reading frame of a starch–degrading α‐amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α‐amylase (BaqA) has no starch‐binding domain, and together with a few putative α‐amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch.

Conclusions

A novel raw starch–degrading B. aquimaris MKSC 6.2 α‐amylase BaqA is proposed to be a member of new GH13 subfamily.

Significance and Impact of the Study

This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.     

Pleiotropic phenotypes of a <Emphasis Type="Italic">Yersinia enterocolitica flhD</Emphasis> mutant include reduced lethality in a chicken embryo model

Background  

The Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.