Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing

Table 1 Construction of mutant PsXRs

Enzyme	Amino Acids^[a]		PCR
Wild-type NT-XR	GPQ SFVE LN	IPKSN TVPR L	Template	Primers^[b]
1-2F7	·········	··S·P·····	NT-XR	5'-TCTTGGGACAGTMNN GGAMNN TGGAATGATGGCAATG-3'
				5'-CATCATTCCANNK TCCNNK ACTGTCCCAAGATTGTTGGAAAAC-3'
2-2C12	·········	··SGP···F·	1-2F7	5'-TTTCCAACAAMNN TGGGACAGTAGGMNN CGATGGAATGA-3'
				5'-CATTCCATCGNNK CCTACTGTCCCANNK TTGTTGGAAAACA-3'
3-4A9	·········	··SGP···F·	2-2C12	5'-CCTTGGTTCAAMNN AACGAAAGAMNN AGGACCGAACGAAGAG-3'
				5'-GTTCGGTCCTNNK TCTTTCGTTNNK TTGAACCAAGGTAGAG-3'

[a] Dots indicate the same amino acid residues as PsXR. Bold letters show the randomized amino acid residues of PsXR in this study. [b] In the degenerate primers, underlines indicate mutated regions; M stands for A or C, K for T or G, and N for A, T, G or C. So in each round of saturation mutagenesis, the theoretical library size was (4*4*2)^2 = 1024.

ISSN: 1475-2859