Chemicals and reagents
TempliPhi100DNA amplification kit was purchased from Roche (Basel, Switzerland). Ortho-nitrophenyl-β-D-galactopyranoside (oNPG), isopropyl thio-β-D-galactoside (IPTG), peptone, and yeast extract were provided from Bio Basic Inc. (Ontario, Canada). ProBon™nickel-chelating resin was supplied by Invitrogen Corp. (Carlsbad, CA, USA). The PCR reagents, restriction endonucleases, T4 DNA ligase and Taq polymerase, PCR primers(IDT, USA) were purchased from Fermentas (Thermo Fisher Scientific Inc., Waltham, USA).
Bacterial strain and expression plasmid
The gene lacA (2061 bp, accession No EU585783) coding for β-galactosidase from Bacillus subtilis strain VTCC-DVN-12-01inserted into the expression vector pET22b(+) resulting in pELacA was described in a previous study . The Escherichia coli strain JM109(DE3) [endA1, recA1, gyrA96, thi, hsdR17 (rk
+), relA1, supE44, λ-, ∆(lac-proAB), [F’, traD36, proAB, lacIqZ∆M15], IDE3] (Promega Corp, Madison, WI) and the plasmid pELacA were used for the expression of the wild-type and mutant β-galactosidase LacA and for screening of LacA mutants. E. coli cells were cultivated in Luria-Bertani (LB) medium containing1% (w/v) bacto tryptone, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl, pH 7–7.5 and 50 μg/ml of ampicillin. LB agar contained additionally 2 % (w/v) agar and 100 μg ampicillin/ml.
Error-prone rolling circle amplification
The recombinant plasmid pELacA was used as a template for the epRCA reaction by using the TempliPhi 100 DNA amplification kit consisting of a sample buffer containing random hexamers that prime DNA synthesis nonspecifically, an enzyme mix containing Φ29 DNA polymerase and a reaction buffer containing deoxyribonucleotides. An amount of 25 pg of pELacA was mixed with 5 μl of sample buffer and heated at 95 °C for 3 min to denature the plasmid, then immediately cooled down to room temperature. The amplification was started by adding 5 μl of reaction buffer, 0.2 μl of enzyme mix and MnCl2 at the final concentration of 1.5 mM and incubated to 30 °C for 24 h. The mixture was heated at 65 °C for 10 min to inactivate the enzyme. The quantity of amplified DNA was estimated on 1 % (w/v) agarose gel electrophoresis and by measuring its absorbance at 260 nm with a spectrophotometer UV-2500 (LaboMed Inc., Culver City, CA, USA).
Construction of mutant libraries
epRCA products were digested with MluI in a mixture contained 12 μl (600 ng) of ep-RCA product, 5 μl 10 × buffer R, 2 μl (20 U) MluI and 31 μl H2O. After 6 h of incubation at 37 °C, the digested products were purified by using the MinElute Reaction Cleanup kit (Qiagen) and ligated with T4 ligase. The ligated epRCA transformed into E. coli JM109(DE3) by using standard electroporation method with a 0.1 cm electrode cuvette under the conditions at 1.8 kV, 200 Ω, and 25 F. Transformed cells were plated onto LB plates containing 50 μg/ml of ampicillin and incubated at 37 °C overnight. Colonies harboring putative mutation sites in lacA were prescreened for a higher β-galactosidase production.
Plasmid DNA isolation was carried out by methods as previously described . DNA sequencing was performed on ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems Inc., Foster City, USA). Sequence alignments constructed and analyzed using the program MegAlign DNAStar. E. coli DH5α and JM109(DE3) cells were transformed using heat shock method that has been previously described .
Screening β-galactosidase activity of mutants
To screen β-galactosidase activity of mutants, the individual transformants carrying the putative mutant lacA gene were randomly selected and grown in 300 μl LB medium containing 100 μg/ml ampicillin in 96-deep well plates at 37 °C overnight with agitation of 250 rpm. 25 μl of overnight culture was transferred from each well to second 96-deep well plates containing 300 μl LB medium containing ampicillin. The culture was cultivated at 37 °C with agitation of 300 rpm until an optical density (OD) at 600 nm of 0.6 to 0.8 was reached (for approximately 4 h), then 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added. The culture continuously incubated at 37 °C with agitation of 300 rpm for 16 h of induction. The cell cultures were used as the enzyme source to screen the activity.
The procedure for measuring β-galactosidase activity of colonies from site-saturation library was performed according to Griffith et al.,  with oNPG substrate (4 mg/ml) in 0.1 M Na-phosphate buffer, pH 7. The absorbance of culture density at 600 nm, reaction mixture at 420 and 550 nm was measured in a microplate reader Elx800™ (BioTek Instruments Inc., Winooski, USA) and β-galactosidase activities were calculate in Miller units following equation: Miller Unit (nM/min/OD
) = [(OD
420 − 1.75 × OD
550)] × V
1/(T × V
2 × OD
600). In that, OD420 and OD550 are read from the reaction mixture; OD420–1.75 × OD550, nmoles formed per milliliter; 1.75 × OD550, light scattering at 420 nm; T, incubation time (min); V1 (ml), total assay volume; V2 (ml), volume of culture used in the assay; OD600 reflects cell density in the washed cell suspension. All measurements were carried out in triplicate with the resulting values being the mean of the cumulative data obtained.
Site-directed and saturation mutagenesis
Site-directed and saturation mutagenesis were performed by a one-step polymerase chain reaction (PCR) method, using plasmid pELacA as template and a pair of mutagenic primer. Randomization codon was performed with a pair of primer that introduced a codon NNK at selected positions. Whole pELacA plasmid was amplified in PCR mix containing 5 μl of 10× PCR buffer, 4 μl of 25 mM MgSO4, 4 μl of 2.5 mM dNTP, 0.5 μl of 2.5 U/μl Pfu polymerase (Thermo), 1 μl of each primer (10 pmol), 1 μl of pELacA (50 ng), and 33.5 μl H2O. The thermocycler conditions were performed as follow: 95 °C/4′; 18 cycles of 95 °C/30″, 54 °C/1′, 72 °C/8′; and 72 °C/10′. Then, 10 U of DpnI restriction enzyme (Thermo) was added the reaction products and incubated for 2 h at 37 °C to digest pELacA template. The PCR products were purified using a PCR purification kit (Thermo). The resultant plasmid DNA was transformed into chemically competent JM109(DE3) cells. Transformants were selected on LB agar containing 100 μg/ml after incubation overnight at 37 °C.
The transformants E. coli JM109(DE3)/pELacA harboring the wild type and mutant lacA gene were cultivated in 5 ml of LB medium containing 5 μl of 100 mg ampicillin/ml at 37 °C with agitation at 220 rpm. Five hundred μl of the overnight culture were transferred into 50 ml of LB medium containing 50 μl of 100 mg ampicillin/ml in a 250-ml Erlenmeyer flask. The culture was cultivated at 37 °C with agitation at 200 rpm until an optical density (OD) at 600 nm of 0.6–0.8 was reached (for approximately 4 h), then 50 μl of 100 mM IPTG was added. The culture was continuously incubated at 37 °C with agitation of 220 rpm for 6 h of induction. Cells were harvested by centrifugation at 5000 rpm for 10 min at 4 °C. Wet cells were used for enzyme purification.
The recombinant LacA fused with a C-terminal 6 × histidine-tag was purified using affinity chromatography with Ni2+-ProBond™ resin under native conditions. An amount of 500 mg wet cells from a 50-ml culture in LB medium was harvested by centrifugation at 4000 rpm and 4 °C for 10 min, washed with 8 ml of water and resuspended in 8 ml of 1× native purification buffer containing 50 mM NaH2PO4, 0.5 mM NaCl,100 mM imidazol, pH 8.0. To the mixture, lysozyme was added at a final concentration 0.5 mg/ml and incubated on ice bath for 30 min. The cell mixture was disintegrated by ultrasonic waves (3× 1 min with 1 min pause). The supernatant of the cell lysate was obtained by centrifugation at 13,000 rpm for 10 min and loaded on to a column containing 2 ml resin, which was equilibrated with native binding buffer and incubated for 45 min at room temperature with gentle hand shaking for several times. The column was washed with 3 times of 8 ml native wash buffer. The bound protein was eluted with 8 ml of 1× native purification buffer containing 250 mM imidazol. The enzyme solution was used for characterization.
Electrophoresis analysis and protein concentration
The homogeneity and molecular mass of the β-galactosidase was determined by 12.5 % SDS polyacrylamide gel electrophoresis  with Biometra equipment (Göttingen, Germany). Proteins were visualized by staining with 0.1 % (w/v) Coomassie Brilliant Blue R-250. Protein concentrations were estimated by the method of Bradford with the bovine serum albumin as standard .
β-Galactosidase activity assay
To estimate the activity of the purified β-galactosidase, 1 μl (3.4 μg) purified enzyme solution was added to 74 μl 22 mM oNPG in 100 mM buffer Z (40 mM Na2HPO4.7H2O, 60 mM NaH2PO4.H2O, 10 mM KCl, 1 mM MgSO4.7H2O, 50 mM 2-Mercaptoethanol) pH 7, incubated at 50 °C for 10 min. Then the reaction was stopped by addition of 25 μl 1 M Na2CO3. The absorbance was read at 420 nm against a blank containing oNPG, buffer Z but without enzyme solution. The following equation was used to calculate units of β-galactosidase activity: U/ml = [OD
420 × V
1]/[0.0045 × 1000 × T × V
2] (ii). In the equation (ii), OD420/0.0045×1000: μmoles what formed per milliliter; T, incubation time (min); V1 (ml), total assay volume; V2 (ml), enzyme volume used in the assay.
pH and temperature dependency of mutant enzymes
The temperature and pH optimum of thepurified wild-type and mutant LacA, 3.4 μg enzyme for each reaction, were determined by measuring the activity as described above using 100 mM buffer Z (pH 7) at the temperature range of 30 to 70 °C and using different buffer pH 4.0–9.0 (100 mM potassium acetate buffer pH 4.0–6.0, 100 mM Na-phosphate buffer pH 6.0–8.0, or 100 mM Tris–HCl buffer pH 8.0–9.0) at 50 °C, respectively, for 5 min.
For the determination of temperature and pH stability, purified enzyme, 3.4 μg protein for each reaction, was incubated in 100 mM buffer Z at different temperatures from 30 to 60 °C for 0–72 h, and in 100 mM Na-phosphate buffer pH 4.0 and 9.0 at 30 °C for 0–72 h, respectively. The remaining activity was then determined. Half-life (T1/2) of each mutant enzyme was determined as follows: T1/2 = ln2/k, where Ut, U0, and k are enzyme activity at t min, initial enzyme activity, and the apparent rate constant, respectively.
All data were averaged and experiments were performed three times. Specific activities were calculated from these averages. The entire assay experiments were than repeat two more times and three specific activity values were averaged. The errors (SD) were calculated by STDEV function of Excel.
Characterization of mutants
The apparent kinetic parameters (Vmax and Km) were determined against 0.1-6 mg/ml of oNPG as a substrate using Lineweaver-Burk plots. Activities were recorded at 55 °C and calculated on the basis of an extinction coefficient for o-nitrophenol of 4500 M−1 cm−1 at 420 nm.
DNA and amino acid sequence analysis
Homologies of the DNA and amino acid sequences were determined with the program Megalign DNAStar.