| Summary: | 博士 === 國立中興大學 === 分子生物學研究所 === 91 === The yellow-pigmented gram-negative bacterium Xanthomonas campestris pv. campestris (Xc17) can produce a water-soluble extracellular polysaccharide (xanthan gum) that is extensively applied in the food and other industries. However, owing to its low -galactosidase activities, Xc17 can’t grow well in the medium containing lactose as the sole carbon source, so it can''t directly use whey as a substrate for xanthan production. A lactose-utilizing strain of X. campestris strain (Xc17L) obtained by nitrous acid mutagenesis can use lactose directly for xanthan gum production without incorporation of any exotic DNA or antibiotic resistance gene is reported here. The results of characteristic analyses of Xc17L, including -galactosidase activity, optimal condition of enzymatic activity, strain stability and xanthan gum production, indicate that Xc17L is potentially useful for xanthan production from whey.
In the genome of Xc17, three annotated -galactosidase genes (galA, galB and galC) are molecularly characterized. Opposite to GalA and GalB which show no detectable enzyme levels even after being cloned in-frame with the lacZ'' gene of vector, GalC displays significant -galactosidase activity. Based on the results of insertional mutation, transcriptional fusion assay and Western blotting, galC is co-transcribed with the upstream gene(s) and expressed constitutively. GalC is a homologue of X. axonopodis pv. manihotis Bga proposed to be involved in cleaving -1,3- and -1,4-linked galactose from the terminal position of polysaccharide chains available in the environment instead of lactose. The homology together with the genome organization suggest that galC is not responsible for lactose utilization, but polysaccharide degradation. Xc17 appears to be deficient in lactose uptake system, because Xc17 carrying cloned lacZY can grow on lactose but not those with cloned galC or lacZ only. Results of DNA sequencing of galA and galB, mutation in galA, galB and galC, and the Western blotting of galC indicate that the galA, galB and galC genes are not correlated to the elevation of -galactosidae activity in Xc17L.
A novel -galactosidase gene, galD, is revealed by insertional mutagenesis, and its homology, galE, exists in the genome. GalD, indeed expressed in Xc17, exists obvious -galactosidase activity, but GalE shows no detectable enzymatic activity. Combined the genome organization and Western blotting, it is indicated that galD is co-transcribed with the upstream two genes. The results of phylogenetic analysis strongly suggest that galD and galE form a separately evolutional divergency which is significantly different from the traditionally well-known GH35 family. Comparing the amino acid sequences of galD and galE in Xc17 and Xc17L, no difference is observed. But, the result of Western blotting indicates that the amount of GalD expressed in Xc17L is as three times as that in Xc17 which shows a perfect consistence between the -galactosidase activity and the expressed amount of GalD. So, elevated -galactosidase activity in Xc17L results from amount of GalD. Using the strategy of affinity chromatography，GalD protein can be purified and a lot of key factors determining the enzyme activity of GalD are revealed. Seven amino acid residues of GalD are substituted by site-directed mutagenesis, separately, and effects of a.a. substitution on the enzyme activity of GalD are evaluated.
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