Quantitative PCR study on the mode of action of oligosaccharide elicitors on penicillin G production by Penicillium chrysogenum.

Aim: To investigate the effects of single and multiple additions of the oligosaccharide elicitors, obtained from alginate and locust bean gum, on penicillin G production and the transcript level of penicillin G biosynthetic genes.

Methods And Results: The transcript copy numbers and penicillin G concentration in liquid cultures of Penicillium chrysogenum grown under control and elicited conditions were compared using quantitative PCR and HPLC assay respectively. An increase in the penicillin G production rate and transcript copy numbers of the three major penicillin G biosynthetic genes pcbAB, pcbC and penDE was observed in the elicited cultures compared to control cultures.

Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved.

Aims: A new strain of Bacillus, Bacillus cereus SPV, was found to be capable of using a wide range of carbon sources for the production of polyhydroxyalkanoates (PHAs) (Valappil et al. 2007b). Limiting nutrient in the culture conditions is crucial for PHA production.

Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterised Bacillus cereus SPV.

A newly characterised Bacillus strain, Bacillus cereus SPV was found to produce PHB at a concentration of 38% of its dry cell weight in shaken flask cultures, using glucose as the main carbon source. Polymer production was then scaled up to 20 L batch fermentations where 29% dry cell weight of PHB was obtained within 48 h. Following this, a simple glucose feeding strategy was developed and the cells accumulated 38% dry cell weight of PHB, an increase in the overall volumetric yield by 31% compared to the batch fermentation.

Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces.

Gram-positive bacteria, notably Bacillus and Streptomyces, have been used extensively in industry. However, these microorganisms have not yet been exploited for the production of the biodegradable polymers, polyhydroxyalkanoates (PHAs). Although PHAs have many potential applications, the cost of production means that medical applications are currently the main area of use.

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp.

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B.

Effects of oligosaccharides and polysaccharides on the generation of reactive oxygen species in different biological systems.

Use of carbohydrates as elicitors is a novel technique for enhancement of the production of industrially important microbial products. The relation between the levels of ROS (reactive oxygen species) and overproduction of antibiotics in microbial cultures has already been established. In the present study, we aimed to exploit the ROS response to develop a fast technique to assess the potential of oligosaccharides [oligoguluronate, oligomannuronate and MO (mannan oligosaccharides)] and polysaccharides [alginate and LBG (locust-bean gum)] as elicitors for overproduction of secondary metabolites in Streptomyces rimosus and Penicillium chrysogenum.

The influence of small oligosaccharides on the immune system.

In this study, oligosaccharides known to enhance the synthesis of penicillin by Penicillium chrysogenum have been presented to human immune cells and their effect measured. In addition a range of commercially available oligosaccharides have been tested. Results obtained indicate that oligosaccharides with a degree of polymerisation greater than 6 and with a tendency to form helical structures are most effective at influencing the immune system as measured by the production of reactive oxidising species.

Elicitor effects on Penicillium chrysogenum morphology in submerged cultures.

Changes in morphology and sporulation were investigated in liquid cultures of Penicillium chrysogenum P2 supplemented with carbohydrate oligosaccharides. Sodium alginate and locust-bean (Ceratonia siliqua) gum-derived oligosaccharides were used as elicitors. Spore germination was inhibited by the addition of OG (oligoguluronate) elicitor (30% inhibition when compared with control).

Elicitor effects on reactive oxygen species in liquid cultures of Penicillium chrysogenum.

Activity of reactive oxygen species (ROS) was investigated in liquid cultures of Penicillium chrysogenum P2 supplemented with carbohydrates. Oligosaccharides lowered the ROS activity in all samples. The greatest effect occurred when oligosaccharides were added to samples 48 h after inoculation.

Purification and biochemical properties of a galactooligosaccharide producing beta-galactosidase from Bullera singularis.

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%.

Elicitation of plants and microbial cell systems.

Plants show physiological and morphological responses to a range of physical and chemical factors known as 'elicitors'. These responses have been considered as defence reactions 'elicited' by the plants' biochemical factory to ensure their survival, persistence and competitiveness. Recently examples have been cited of elicitation in some fungal and bacterial cultures.

Using 2', 7'-dichlorodihydrofluorescein-diacetate to assess polysaccharides as immunomodulating agents.

Neutrophils play a key role in the defense against microbial infections. One of their primary weapons to destroy microbes is the production of reactive oxygen species (ROS). This paper shows how 2', 7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) was used to measure the effects of polysaccharides on the production of ROS by polymorphonuclear leukocytes (PMNs).

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase.

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors.

Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on cgtase.

Cyclodextrin glycosyltransferase (EC 2.4.1.

The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977).

Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days.

Enhanced penicillin production by oligosaccharides from batch cultures of Penicillium chrysogenum in stirred-tank reactors.

Alginate and galactomannan-derived oligosaccharides enhanced the production of penicillin G when added to stirred tank reactor cultures of Penicillium chrysogenum. The addition of oligomannuronate and oligoguluronate blocks increased penicillin G yield by 47% and 49%, respectively. The effect of mannan oligosaccharides was found to be more pronounced with 69% higher yield than the control cultures.

Separation, characterization, and specificity of alpha-mannosidases from Vigna umbellata.

Information about the specificity of glycosidase enzymes is important since it affects their use for characterization and synthesis of oligosaccharides. Two alpha-mannosidases (EC 3.2.

Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum.

Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated.

Use of INT to determine the respiratory activity of immobilised and free Penicillium chrysogenum.

The specific oxygen uptake rates (OUR) of kappa-carrageenan-immobilised and free cell cultures of Penicillium chrysogenum were determined using an oxygen electrode in a closed chamber. This was compared with the respiratory activity determined by the extent of staining with iodo-nitrophenyl tetrazolium chloride (INT). The degree of INT staining correlated with the OUR; an increase in INT deposition corresponding to an increase in the measured OUR.

Primary sequence analysis and representation techniques in carbohydrates.

Sequence similarity calculations of carbohydrates present several problems which must be addressed if a computer implementation is to be achieved. These problems range from the computational representation of the complex carbohydrate structure to the method by which the comparison of residue and linkage is to be made. This paper therefore discusses the form of this representation and how two or more carbohydrates can be meaningful compared.

Physiological studies related to the immobilization of Penicillium chrysogenum and penicillin production.

Using the production of penicillin by Penicillium chrysogenum as a model system, certain physiological aspects of immobilized and free cell cultures were compared. Reducing the immobilized viable spore loading (from 4 x 10(4) to 2 x 10(3) spores ml-1 gel) and initial bead diameter (from 3.5-4.

The application of aqueous two-phase systems to oligosaccharide synthesis by alpha-mannosidase catalysed glycosyl transfer reactions.

Aqueous two-phase systems formed from PEG and dextran have been applied to the synthesis of oligosaccharide by Jack bean alpha-mannosidase in reverse. Whilst rates of synthesis and percentage yields were similar in two-phase systems and one-phase aqueous buffer systems, a ten-fold increase in yield of product per unit of enzyme was seen. In addition, the use of aqueous two-phase systems offers potential process advantages over one-phase systems for the synthesis of oligosaccharides.

The effects of spore loading on the growth of Penicillium chrysogenum immobilised in kappa-carrageenan.

Penicillium chrysogenum spores were immobilised in kappa-carrageenan. The effect of the number of viable spores immobilised per bead on the rate of germination and degree of subsequent mycelial growth was investigated. The distribution of active mycelium throughout the bead was determined.