Christopher Bucke

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.

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.

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. In this study, B. cereus SPV was grown in different culture conditions with limitation of potassium, nitrogen, sulphur and phosphorous to establish the impact of nutritional limitation on PHA production.

Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase : Solvent effects on CGTase

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. Addition of small amounts (10% v/v) of polar organic solvents can affect both the overall production yield and the type of cyclodextrin produced from a maltodextrin substrate under simulated industrial process conditions. Using CGTase from Thermoanaerobacter sp. all solvents produced an increase in cyclodextrin yield when compared with a control, the greatest increase being obtained with addition of ethanol (26%). In addition product selectivity was affected by the nature of the organic solvent used: beta-cyclodextrin was favoured in the absence of any solvent and on the addition of dimethylsulphoxide, t-butanol and dimethylformanide while alpha-cyclodextrin was favoured by addition of acetonitrile, ethanol and tetrahydrofuran. With CGTase from Bacillus circulans strain 251 relatively smaller increases in overall cyclodextrin production were achieved (between 5-10%). Addition of t-butanol to a B. circulans catalysed reaction however did produce the largest selectivity for beta-cyclodextrin of any solvent-enzyme combination (82%). The effect of solvent addition was shown not to be related to the product inhibition of CGTase, but may be related to reduced competition from the intermolecular transglycosylation reaction that causes degradation of cyclodextrin products. This rate of this reaction was shown to be dependent on the nature of the organic solvent used.

Enhancement of glucose oxidase production by Penicillium variabile P16

Effects of the polysaccharides alginate and locust bean gum, and oligosaccharides oligomannuronate (OM) and oligoguluronate (OG), on glucose oxidase (GOD) production by Penicillium variabile P16 were studied. Small increases were observed when the cultures were supplemented with OG and OM blocks with an average degree of polymerization (DP) of approximately ten. With 200 mg l−1 OM blocks addition at 0 h, the increase reached 32.1% compared with the control; however, regardless of the time of addition, large increases (up to approximately 70%) in GOD production were obtained with 100 and, particularly, 200 mg l−1 of alginate-derived oligosaccharides (OG and/or OM blocks) with a DP of approximately seven. No significant influence was observed on mycelial biomass.

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. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 microg/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. (c) 1997 John Wiley & Sons, Inc.

Mycelium-bound lipase from a locally isolated strain of Aspergillus flavus link: Pattern and factors involved in its production

Aspergillus flavus produces a lipase (EC 3.1.1.3) which is partly bound to the mycelium during growth. The production of the mycelium-bound lipase is concomitant with growth, and declines when growth ceases. Maximum productivity of the enzyme is obtained when the culture is incubated at 30°C, an initial culture pH of 6·5 and with 2% (w/v) each of corn oil and yeast extract as carbon and organic nitrogen source. Yeast extract affects not only the production of lipase but also the secretion of proteases into the culture medium. Production of the latter enzymes, which inactivate the free lipase, is enhanced by adding yeast extract (1–2%, w/v) to the culture medium. However, at 5% (w/v) yeast extract concentration, proteolytic activity is not detected and consequently, the activity of free lipase may easily be measured. Free lipase activity can easily be detected when 0·001 mol dm−3 EDTA is added to the culture medium. The presence of the chelating agent enhances the production and maintains the stability of the extracted mycelium-bound lipase.

Enzymatic Synthesis of Oligosaccharides

The aim of this research is to develop a model to describe oligosaccharide synthesis and simultaneously lactose hydrolysis. Model A (engineering approach) and model B (biochemical approach) were used to describe the data obtained in batch experiments with (J-galactosidase from Bacillus circulans at various initial lactose concentrations (0.19-0.59 mol/kg). A procedure was developed to fit the model parameters and to select the most suitable model. The procedure can also be used for other kinetically controlled reactions. Each experiment was considered as an independent estimation of the model parameters, and consequently, model parameters were fitted to each experiment separately. Estimation of the parameters per experiment preserved the time dependence of the measurements and yielded independent sets of parameters. The next step was to study by ordinary regression methods whether parameters were constant under the altering conditions examined. Throughout all experiments, the parameters of model B did not show a trend upon the initial lactose concentration when inhibition was included. Therefore model B, a galactosyl-enzyme complex based model, was chosen to describe the oligosaccharide synthesis, and one parameter set was determined for various initial lactose concentrations. This chapter has been published as: Boon MA, Janssen AEM, van der Padt A. 1999. Modelling and parameter estimation of the enzymatic synthesis of oligosaccharides by |3-galactosidase from Bacillus circulans. Biotechnol Bioeng 64:558-567.

Substrate preference of mycelium-bound lipase from a strain of Aspergillus Flavus Link

Aspergillus flavus mycelium-bound lipase demonstrates high preference towards short chain triacylglycerols and discriminates against triunsaturated triacylglycerols e.g. triolein. The great discriminating power of its lipase against triolein was shown in comparison with its ability to catalyse the hydrolysis of shorter chain triacylglycerols e.g. tricaprin and less was shown when hydrolysing tripalmitin. A similar phenomenon was noted when the mycelium-bound lipase was used to catalyse the reaction of coconut oil with palmitic acid or oleic acid in n-hexane. The relative percentages of octanoic acid and decanoic acid of coconut oil remaining after 20 h reaction were much less than those of the medium, long and unsaturated chain fatty acids suggesting that short chain fatty acids are preferred. The lipase hydrolyses coconut oil faster than palm olein followed by corn oil, rapeseed oil, soy bean oil and cottonseed oil. This indicates that A. flavus lipase has preference for oils containing saturated fatty acids rather than unsaturated fatty acids.

α-Mannosidase-catalysed synthesis of novel manno-, lyxo-, and heteromanno-oligosaccharides: A comparison of kinetically and thermodynamically mediated approaches

Abstract The α-mannosidase from jack bean has been used to catalyse the production of a range of novel oligosaccharides and oligosaccharide derivatives. p-Nitrophenyl oligosaccharide glycosides have been synthesized by glycosyl transfer from p-nitrophenyl α-mannoside donor to a range of p-nitrophenyl glycoside acceptors. Oligosaccharides have also been synthesized by the condensation of mannose with a range of mono- and oligosaccharide acceptors. The structures of oligosaccharides formed from mannose by condensation have been investigated by methylation analysis and NMR spectroscopy.