Optimisation of production of extracellular non-haem peroxidases by Thermomonospora fusca BD25 in aerobic bio-reactor conditions.

Peroxidases are involved in redox reactions [l-2] and in particular the redox reaction of haem-containing horseradish peroxidase has been exploited in a diagnostic procedure such as serum cholmterol determination 131. The current chemical techniques for the determination of serum cholesterol is subjected to various interference 14-61, The use of biological catalysts such as horseradish peroxidase has substantially improved the results [7]. However, as horseradish peroxidase contains labile haem, it i s subjected to denaturation by temperature and could give rise to falsely low results. The problem can be circumvented by using a thermostable peroxidase enzyme such as Thermomonospora .fusca BD25 peroxidase 191. However, the availability of this peroxidases is limited, since the enzyme is expressed to a very low level by T fusca BD25 and detection in the extra-cellular supernatant is often difficult [lo]. The problem can be over come by optimisation of the growth conditions of T fusca BD25 [ 1 I]. However, in-house optimisation dose not allow precise monitoring of the growth parameters such as dissolved oxygen, temperature and pH. In this respect, the use of an automated bio-reactor would be helpful to resolve this problem. In this paper we describe the optimum bio-reactor conditions for the production of extra-cellular non-haem peroxidases. Stock cultures of T fuscrr BD25 were maintained as a suspension of spores and hyphal fragments in 20 YO ( viv) glycerol at -20 "C and routinely cultured on L-agar plates or slants [ 121 with subsequent incubation at 45 "C for 48-72 hours or until sporulation had occurred. For peroxidase production in an automated bio-reactor suspensions, 50 ml (48 h old) liquid cultures of T. fusca BD25 was inoculated directly into a sterilise production medium ( I L) based on that described by Ramachandra ef al. [ 131. The medium contained 8.0 g oat spelt xylan (Sigma); 6.0 g yeast extract (Oxoid); 0.1 g (NH4)2 SO4; 0.3 g NaCI; 0.1 g MgS047H20; 0.02 g CaC03; 500 pl antifoam (204); 100 mM potassium phosphate buffer and 1 ml of trace element solution per litre of distilled-water, final pH of 7.5. The trace element solution contained 1.0 g FeS047H20; 0.9 g ZnS047H20; 0.2 g MnS047H20 per litre of distilledwater. Inoculated cultures were incubated at 50 "C at 250 rpm with dissolved oxygen of either 50 % (v/v) or 5 YO (viv). The growth parameters during the exponential growth phase were carefully monitored an any deviations from the setting values automatically corrected. The results of optimisation of the production of extra-cellular non-haem T fusca BD25 peroxidases using an automated bioreactor are shown in Figure I . It should be noted from the graph that maximum peroxidase production (0.1 Uml-') occurred after 36 h of incubation at 50 YO (v/v) dissolved oxygen at a pH of 8.41; temperature 50 "C and agitation 250 rpni. The level of enzyme production, however, falls rapidly after 42 h of incubation, reaching a minimum level at 60 h. The peroxidase level obtained from the bio-reactor experiment corresponds approximately to an increase of three fold with respect to in-house production level. Thus, the use of automated bio-reactor has reduced the time of production of this enzyme and significantly increased the expression level.