Normas y Leyes a Utilizar

The activity of acyl-CoA hydrolase was not determined in the ultracentrifuged and dialysed samples, so it was not possible to estimate the yield of acyl-CoA hydrolase activity in the various eluted samples. The presence of mercaptoethanol in the buffer prevented the use of the specific acyl-Co A hydrolase assay (using DTNB) to track the elution of the enzyme. Instead, non-specific esterase assays were used (Section 2.5.16).

C h a p te r 4. E n zym o lo g y o f lip id d e g ra d a tio n in S. liv id an s

Fraction Esterase activity Protein concentration

(nmol min-^mg i) +(SEM) (mg ml-i)

wash 1 13.3 (0.56) 22.2

wash 2 2.8 (0.09) 6.3

200mM fraction 118 (1.7) 0.47

Table 4.16.4 Non-specific esterase activity o f the eluted fractions, using a 4-nitrophenol acetate substrate.

No other eluted fractions showed esterase activity. Some activity eluted in the first two wash fractions and in the 200mM fraction. This could have been due to either acyl-CoA hydrolase activity or another type of esterase. The esterase activity in the 200mM fraction may come from the 12 kDa contaminating protein, but alternatively it could be integral to the multifunctional protein subunits. Specific acyl-CoA hydrolase activity was found on

the mutifunctional complex of E. coli (personal communication - K. Bartlett) . It is

possible that the inducible thioesterase activity in E. coli (Samuel and Ailhaud, 1969) is

due to the acyl-CoA hydrolase found on the p-oxidation enzyme complex, since both activities are induced by growth on oleate. It would be interesting to determine the substrate specificity of the purified acyl-CoA hydrolase in order to compare it with the data obtained on activity in the CFE (Section 4.14.2) as a method of determining if there

is more than one acyl-CoA hydrolase activity in S. lividans. That could not be done in

this study due to time constraints.

The function of acyl-Co A hydrolase in cells is open to debate, especially since it seems that at least some of the activity originates from the p-oxidation multifunctional enzyme, an unlikely location since acyl-CoA intermediates are catabolized on the complex. Long- chain acyl-CoAs have a detergent effect in the cell, so a regulation of long-chain acyl-CoA esters by acyl-CoA hydrolase activity could be occurring. This is supported by the preference of the enzyme(s) for long-chain substrates. Regulation of acyl-CoA hydrolase activity could occur by the level of CoASH in the cell. When this is low, the hydrolase would operate optimally, since product inhibition could not occur. When CoASH was high, the acyl-CoA synthetase and the P-oxidation cycle activities would be favoured. However, inhibitory concentrations of CoASH to the acyl-CoA hydrolase were not

determined, nor was the of CoASH for acyl-CoA synthetase (partly because the

enzyme could not be purified). Functions of the acyl-CoA hydrolase in lipid biosynthesis must also be considered, since the selection of a certain length of fatty acid would be facilitated if the CoA was removed when the carbon chain grew to a certain length. This is further discussed in Section 1.5.5.

4.17 Summary

The data obtained in this chapter provides a pattern of overall lipid catabolism in an actinomycete, something which has not been done before. Lipase activity was reasonably

C h a p te r 4. E n zym o lo g y o f lip id d e g ra d a tio n in S. lividans

low, especially compared with other organisms. However, it is not clear that the lipase activity being monitored comprised aU of such activity occurring in the medium. As previously stated there could have been free rather than cell-associated lipases present in the culture broth.

The component enzymes of the p-oxidation pathway were identified in S. lividans, which

was found to have a system resembling that of E. coli. The use of an in vitro system was

not ideal, since it can never be asserted that what is happening in the cuvette is a reflection of the situation in the intact cell. The levels of enzyme activities were assayed, though, which provided an estimate of activity in the true cell system. Levels of p-oxidation enzymes were low compared to other organisms, but the whole pathway enzyme assays had comparable activities that were not particularly improved by the addition of external

enzymes to boost specific reaction steps. The acyl-CoA synthetase may be mainly

membrane-bound, so amounts of enzyme in the cell free extract may be very low compared to those in the cell. It was not possible to test the K^s of the enzymes not purified in the column process, since interactions with inhibitory (and promoting) enzymes and their metabolites could influence the rates. The affinity of an enzyme for a particular intermediate cannot therefore be compared with another. This study did not employ analogous substrates for each of the pathway steps, since these intermediates require chemical synthesis that was beyond the scope of the project.

Another problem with estimating the effects of various enzymes on overall flux is the use of fatty acids in a solution of DMSO to test the activity of the acyl-Co A synthetase. The rate of this enzyme was much less than that of the rest of the pathway, when tested in similar circumstances. The solubility and accessibility of the substrate could have much to do with that. The addition of fatty acids as a complex with BSA improved rates a little, but there is no information on the form in which the fatty acids are transported within the

C h a p te r 5. 2 0 L fe rm e n ta tio n s o f S. lividans

5. 20 L FERMENTATIONS OF STREPTOM YCES U V ID A N S TK24

20 L fermentations were planned to determine the effect of oils on growth in defined media at pilot-scale and also to generate enough biomass to monitor p-oxidation activities at very early points in the growth curve. The use of pilot-scale equipment allowed oxygen uptake, carbon dioxide evolution and respiratory quotients to be determined throughout the fermentation, giving an indication of the type of metabolism occurring in the cells. The acid and base requirements in the cultures could also be investigated.

Conditions for growth were determined from a few initial experiments. Air flow was held at 1 w m since this provided enough oxygen to keep the dissolved oxygen tension (DOT) above 50% even with a high biomass of cells. Stirrer speeds were kept at 500 rpm throughout the experiment. The level of shear produced by this speed was not high enough to prevent pellet formation, but allowed the adequate mixing of fermenter contents and an even distribution of air without damaging the cell mycelia. Due to the volumes of broth that had to be removed from the fermenter at each cell harvesting point, the level of liquid in the fermenter was decreased from an inital 16 L, the maximum running volume for the 20 L fermenter, to above 8 L, which was the lowest volume that did not expose the DOT and pH probes to the air.

The type of inoculum used to seed the fermenter had an effect on the course of a fermentation. Growth proceeded much faster and with a greater density if the seed flask was inoculated with fresh spores rather than the spore stocks (Section 2.1) stored at -70°C and used for the rest of the study. With large-scale fermentations, a high biomass seed culture is necessary to prevent a long lag phase while cell numbers increase.

Therefore each seed flask was inoculated with the spores from S. lividans colonies on half

a densely populated agar plate. The use of fresh plates for each fermentation potentially introduced variation between the individual fermentation cultures, but the same spore stock was used for all the plates to keep variation and mutation to a minimum. It was found that if older, highly pelleted seed cultures were used to inoculate the vessel, the biomass in the fermenter did not increase as rapidly as when younger cultures were used.

The pellets of S. lividans did not break up in the fermenter conditions used, restricting

new growth mainly to the outer surface of existing pellets. If small, but numerous pellets were added as the seed, the potential sites for new growth were much higher. Such a seed culture was prepared with inoculation of a high number of fresh spores followed by incubation for only 24 to 27 h (Section 2.4.3).

The 20 L fermentations were carried out in order that comparisons could be made between the cultures grown on different medium components. Therefore the results of each fermentation are reported in separate sections, followed by a discussion section comparing the observed effects. At least two fermentations were carried out for each type of medium; representative results are given in each case.

C h apters. 20 L fermentations o f S. lividans

Figure 5.1 Photograph of the 20 L fermenter containing a glucose and triolein carbon source culture, 50 h old. The purple colouration of the actinorhodin can clearly be seen in the broth, while some of the cell pellets adhering to the top of the vessel have become blue.