MOLECULAR CLONING AND IN-VITRO MUTAGENESIS OF BACILLUS SUBTILIS XYLOSE ISOMERASE GENE

Abstract

Bacillus subtilis chromosomal DNA was restricted with BamHI to completion and a mini plasmid library was constructed in the plasmid vector, pAT153 also restricted with BamHI. A complete digestion of the chromosomal DNA was performed as it was known that there are no BamHI sites within the Bacillus subtilis xylose isomerase structural gene and its transcriptional regulatory elements. The E.coli host used was DH1 (r-m+) in order to modify heterologus DNA for further manipulations in E.coli. The recombinant plasmids from this library were transformed into a mutant, SPl0 and plated on minimal plates with xylose as sole carbon source. This nutritional selection will therefore yield transformants containing Bacillus xylose isomerase gene contained in the plasmid because it was known that B.subtilis xylose isomerase structural gene does not recombine with the xylose isomerase of E.coli. The recombinant plasmids isolated from 15 xyl+ transformants were found to be similar in size and carried a similar insert of 5.8kb. One of the plasmid, named pTJ1 was characterised by restriction mapping and was found to match the restriction map reported by other workers. pTJ1 was also able to transform other xy1 mutants to xy1+ phenotype. Enzyme assay carried out on crude cell free extracts of E.coli cells carrying the plasmid showed a low activity for xylose isomerase. In order to increase the activity the xylose isomerase structural gene was subcloned into a prokaryotic expression vector, pKK223-3, down stream of the tac promoter. This construct, pTJ18 gave a four fold increase in enzyme activity and therefore was used in mutagenesis studies. Two areas of extensive homolgy at the amino acid level between the xylose isomerase from E.coli, Ampullariella and B.subtilis were identified. Thus it can be inferred that these two regions could be involved in catalysis or binding of the substrate. In order to further narrow down the amino acids involved in isomerisation, a hydrophilicity plot of B.subtilis xylose isomerase was done and it showed that glutamate 238 to have the highest hydrophilicity. Glutamate238 was also located in one of the regions of homology. Therefore, this amino acid may catalyse the reaction by abstracting proton from xylose C-2 and transferring to C-1 to form xylulose in an analogous manner to Glutamate165 in triose phosphate isomerase. Site directed mutagenesis was carried out on a 300bp PstI-XbaI fragment of the xylose isomerase gene subcloned into M13mp19 using an oligonucleotide which could change glutamate to aspartate, another amino acid which is similar in structure but does not abstract proton as efficiently as glutamate. The mutant fragment after confirmation by DNA sequencing was introduced into the gene in plasmid pTJ18. This plasmid pTJ18S1 was unable to complement SP10 cells and enzyme assays carried out using cell free extracts of E.coli carrying this plasmid showed a complete loss in activity. The change therefore has resulted in the synthesis of an inactive polypeptide. In comparison with triose phosphate isomerase the amino acids surrounding glutamate238 in xylose isomerase were then changed to those of E.coli triose phosphate isomerase. This multiple mutant was also unable to show both nutritional complementation with xy1 mutants and any enzyme activities for both glucose and xylose isomerases. These results indicate that glutamate238 could have a role in xylose and glucose isomerisation. Lysine133 was postulated to have a similar role as lysine13 of triose phosphate isomerase which is known to stabilise the reaction intermediate. Site directed mutagenesis using an oligonucleotide to mutate lysine133 to arginine another basic amino acid. This mutation was considered as a means of increasing the stability of the reaction intermediate, cisendiolate The mutant fragment when introduced into the gene in pTJ18 was able to nutritionally complement SP10 cells. Enzyme activities were also detectable. The Km and Vmax for both enzymes xylose and glucose isomerases were therefore determined. The Vmax of ylose isomerase was slightly increased from 0.28 U/mg to 0.33 U/mg while the Km remained unchanged at 0.0135M for both mutant and wild type. The Vmax of glucose isomerase remained unchanged at 0.09 U/mg. However the Km was decreased slightly from 0.28M to 0.21M (mutant). The change being not substantial for both Km and Vmax it could be interpreted that lysine133 may not play a central role in stabilising the intermediate in isomerisation reaction.