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. Gram-negative bacteria, currently the only commercial source of PHAs, have lipopolysaccharides (LPS) which co-purify with the PHAs and cause immunogenic reactions. On the other hand, Gram- positive bacteria lack LPS, a positive feature which justifies intensive investigation into their production of PHAs. This review summarizes currently available knowledge on PHA production by Gram- positive bacteria especially Bacillus and Streptomyces. We hope that this will form the basis of further research into developing either or both as a source of PHAs for medical applications.

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. We have also investigated changes in the production of ROS in neutrophils as a result of the action of the same elicitors. LBG-derived oligosaccharides (MO) were most potent inhibitors of ROS in all systems investigated. This correlates with overproduction of secondary metabolites in microbes and enhancement of a number of mammalian systems. We believe that the effects of oligosaccharides and polysaccharides on ROS production by mammalian and microbial cells can be correlated predicatively with overproduction. The underlying methodology offers a fast screening of elicitors that can be applied across the different systems.

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. Laminariheptaose has been shown to increase reactive oxidising species production by up to 25%, whilst mannan-oligosaccharides with a DP of 6 to 7 decrease production by up to 44%. These and other results show that the immune system can recognise subtle differences in oligosaccharides and that these oligosaccharides could potentially be used to modulate the immune response.

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). Addition of any of the elicitors to stirred-tank cultures increased hyphal-tip numbers, clump area and spore counts. MO (mannan oligosaccharide) had the greatest effect on the parameters studied, followed by OM (oligomannuronate) and OG. Hyphal-tip numbers increased by 19, 29 and 47% with OG, OM and MO respectively. Average clump area in the presence of OG, OM and MO increased by 23, 32 and 59% respectively. A notable increase in spore numbers was observed in all the supplemented stirred-tank reactor cultures, with MO showing the highest enhancement.

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. Through a chronological survey, this Review considers examples of elicitors and elicitation and describes suggested mechanisms of elicitation in plants and microbial cell cultures. The majority of research in this field has been carried out on the plant systems using complex (undefined) biotic elicitors. Carbohydrates are the main class of compounds used as defined elicitors. This Review focuses on carbohydrates as compounds initiating a defence response in cell cultures. Physiological changes brought about on the plant and microbial cultures include expression of novel metabolites and overproduction of already known products. Recent reports confirming elicitation in microbial cultures are of potential importance, as the relative ease of fermentation and scale-up could open an opportunity for the introduction of useful novel metabolites as well as enhancement of commercially useful bioproducts. In this context, a sound knowledge of the elicitor molecules' structure-function relationships and mechanisms of elicitation is essential.

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%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.