Improved thermostability of a Bacillus subtilis
esterase by domain exchange
Markus G. Gall, Alberto Nobili, Ioannis V. Pavlidis, Uwe T. Bornscheuer*
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry,
Greifswald University, Greifswald, Germany
*
Correspondence: U. T. Bornscheuer, Department of Biotechnology and Enzyme
Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Strasse
4, D-17487 Greifswald, Germany. Tel: �+49-3834-864367 Fax: �+49-3834-86794367
E-mail: uwe.bornscheuer@uni-greifswald.de
Experimental details for the mutagenesis experiments
Primers
The following primers were used for incorporation of the BsteE cap domain in the
BsubE gene:
 Primer a (FW): 5’-CAT-CAT-CAC-GTT-CAT-CTT-TCC-CTG-GTT-GCC-AATGG-3’
 Primer b (RV): 5’-CGC TTT TTA TAC TCG CGC GCA TAC TCA AGA ACG
CCT TGA TAC ATG ACC-3’
 Primer c (FW): 5’-GTT CAA GCA GAC GCC GAT GAA GAC CCT CAA GGC
GCT GC-3’
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 Primer d (RV): 5’-CCG CCA GGC AAA TTC TGT TTT ATC AGA CCG CTT
CTG CGT TCT GAT TTA ATC-3’
 Primer e (FW): 5’-GGT CAT GTA TCA AGG CGT TCT TGA GTA TGC GCG
CGA GTA TAA AAA GCG-3’
 Primer f (RV): 5’-GCA GCG CCT TGA GGG TCT TCA TCG GCG TCT GCT
TGA AC-3’
The following primers were used for the preparation of single and double mutants of
BsubE and of the cap mutant (the numbering of the positions refers to the numbering
of amino acid positions in BsubE):
 T7P_fw: 5’-GAG CAT GAA AGT TGT GCC ACC AAA ACC-3’
 T7P_rv: 5’-GGT TTT GGT GGC ACA ACT TTC ATG CTC-3’
 K18R_fw: 5’-GGC GGA GAC CGC GCG GTG CTT TTG C-3’
 K18R_rv: 5’-GCA AAA GCA CCG CGC GGT CTC CGC C-3’
 S88K_fw: 5’-GGT TAT GAG AAA ATT GCT GCC TGC GGA C-3’
 S88K_rv: 5’-GTC CGC AGG CAG CAA TTT TCT CAT AAC C-3’
 K111E_fw: 5’-CAC TGT ACC CAT AGA AGG AAT TGT CC-3’
 K111E_rv: 5’-GGA CAA TTC CTT CTA TGG GTA CAG TG-3’
Experimental details for domain exchange PCRs
According to the experimental scheme presented in the manuscript, PCR 1 is
conducted with primers a and b, PCR 2 with primers c and d, PCR 3 with primers e
and f to amplify the N- and C- termini of BsubE and the cap domain of BsteE, while
in PCR 4 primers a and d were used to combine the fragments of the first three
reactions.
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For PCRs 1-3, 100 µL of master mix were prepared for each one, composed of 77 µL
deionized water, 10 µL 10x reaction buffer, 5 µL DMSO, 2 µL plasmid DNA
template (1:10 dilution of fresh plasmid isolation; for PCR 1 and 2 BsubE-plasmid
and for PCR 3 BsteE-plasmid were used), 2 µL forward primer (12.5 pmol/µL), 2 µL
reverse primer (12.5 pmol/µL), 2 µL dNTP mix (each dNTP 10 mM) and 0.6 µL Pfu
Plus! DNA Polymerase (roboklon, Berlin, Germany). Samples were aliquoted in
25 µL aliquots and PCR was performed with different annealing temperatures. The
reaction conditions were: 1) 95°C for 3 min, 2) 18 cycles: 95°C for 30 s, 50 or 60°C
for 30 s, 72°C for 30 s, 3) 72°C 2 min. Afterwards products were checked by agarose
gel electrophoresis. PCR samples were then purified via preparative agarose gel
electrophoresis (2% agarose gel) and the MinElute® Gel Extraction Kit (Qiagen,
Hilden, Germany). Purification success was proven by another agarose gel.
For PCR 4 the protocol was different due to the fact that no plasmid was used as
template, but the fragments from PCRs 1-3. For 100 µL master mix 79 µL deionized
water, 10 µL 10x reaction buffer, 1 µL DNA purified from PCR 1, 2 µL DNA
purified from PCR 2, 2 µL DNA purified from PCR 3, 2 µL primer a (12.5 pmol/µL),
2 µL primer d (12.5 pmol/µL), 2 µL dNTP mix (each dNTP 10 mM) and 0.6 µL Pfu
Plus! DNA Polymerase were added. The reaction conditions were: 1) 95°C for 3 min,
2) 18 cycles: 95°C for 30 s, 50 or 60°C for 30 s, 72°C for 60 s, 3) 72°C for 3 min.
Afterwards products were checked by agarose gel electrophoresis. PCR samples were
then purified via preparative agarose gel electrophoresis (2% agarose gel) and the
MinElute® Gel Extraction Kit. Purification success was proven by another agarose
gel.
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PCR methods for single and double mutants
Single mutants were prepared for positions 7 and 18. Positions 88 and 111 were
combined in a double mutant, as they seem to contribute to hydrogen bonding. The
mutants were prepared by QuikChangeTM PCR, using as template BsubE or the cap
mutant. The following reaction mixture was prepared for the subsequent
QuikChangeTM PCRs: distilled water (100 μL), 10x Pfu Plus! Buffer (12.5 μL),
dNTPs (2.5 μL, 10 mM each), plasmid pGASTON containing the gene encoding
either BsubE or the cap mutant (2.5 μL, 10 ng/μL), forward primer (2.5 μL, 10 μM),
reverse primer (2.5 μL, 10 μM), DMSO (2.5 μL) and Pfu Plus! DNA polymerase (1.0
μL, 5 U/μL). The reaction was performed using the following thermocycling
conditions: 1) 95°C for 5 min, 2) 20 cycles: 95°C for 30 s, 50 to 65°C for 30 s, 72°C
for 210 s, 3) 72°C for 5 min. The reaction with the best product concentration (as
observed by agarose gel electrophoresis) was used for further experiments. For the
double mutant S88K/K111E the single mutant S88K was first prepared, and then its
DNA sample was used as template for the second mutation (K111E).
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