Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas (Entcheva et al. 2001), Arthrobacter and Rhodococcus (Borneman et al. 1996) and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA from them extracted. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.3

The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli. The gene product of CYP175A1 added hydroxyl groups to both beta rings of beta-carotene to form zeaxanthin (beta,beta-carotene-3,3'-diol) in E. coli, which produces beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. In addition, spectroscopic methods revealed that E. coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone. The predicted amino acid sequence of the enzyme from T. thermophilus does not show substantial similarity with other known beta-carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3). It is concluded that CYP175 A1 represents a new type of beta-carotene hydroxylase of the P450 superfamily.

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