3.1 Sequence analysis of PulAR encoding gene
The pullulanase PulAR gene (GenBank accession number KY273924.1) has a putative translational start site GTG, is 2,259 bp long, has a G+C content of 78.4%, and encodes an enzyme with a predicted molecular mass of 85.0 kDa with a theoretical pI of 5.49. The structure of PulAR was constructed based on the crystal structure of the pullulanase PulA from Anoxybacillus sp. LM18-11 (PDB ID 3WDH), with which it shares the identity 58.29% (Fig. 1A). The structure and protein sequence of type I pullulanase from Anoxybacillus sp. LM18-11 (PulA) (Xu et al. 2014) and that of PulAR were compared. For PulA, D413, E442, and D526 should be nucleophile, acid/base, and transition-state stabilizer, respectively. Accordingly, the corresponding residues D435, E464, and D554 of PulAR are deduced as the catalytic residues. Analysis of the protein sequence of PulAR by NCBI BLASTp showed that it contains the YNWGYDP motif and four conserved regions (I–IV) (Figure S2), which are similar to those of type I pullulanases. No signal peptide was found in the pullulanase PulAR through analysis by Signal P (https://services.healthtech.dtu.dk/service.php?SignalP-4.1). Comparisons with the pullulanase sequences in the GenBank database, listed in Table 1, reveal that PulAR shares 70.8%, 60.2%, 58.29%, 46.48%, 43.59%, 41.5%, 41.14% and 38.14% identity with the thermostable pullulanases from Bacillus stearothermophilus (Kuriki et al. 1990), Geobacillus thermoleovorans (Zouari Ayadi et al. 2008), Anoxybacillus sp. LM18-11 (Xu et al. 2014), Bacillus sp. CICIM 263 (Li et al. 2012), Anaerobranca gottschalkii (Bertoldo et al. 2004), Fervidobacterium pennivorans DSM 9078 (Bertoldo et al. 1999), Thermotoga neapolitana (Kang et al. 2011), and Caldicellulosiruptor saccharolyticus (Albertson et al. 1997), respectively.
Table 1
Comparison of optimum temperatures between bacterial type I pullulanases.
Bacterial source
|
Accession number
|
Optimum temperature
|
Similarity with PulAR
|
Reference
|
Bacillus stearothermophilus
|
1808262A
|
60℃
|
70.8%
|
Kuriki et al. 1990
|
Geobacillus thermoleovorans
|
CAC85704.1
|
70℃
|
60.2%
|
Zouari Ayadi et al. 2008
|
Anoxybacillus sp. LM18-11
|
AEW23439.1
|
60℃
|
58.3%
|
Xu et al. 2014
|
Bacillus sp. CICIM 263
|
AGA03915.1
|
70℃
|
46.5%
|
Li et al. 2012
|
Anaerobranca gottschalkii
|
AAS47565.1
|
65-70℃
|
43.6%
|
Bertoldo et al. 2004
|
Fervidobacterium pennivorans DSM 9078
|
AAD30387.1
|
80℃
|
41.5%
|
Bertoldo et al. 1999
|
Thermotoga neapolitana
|
ACN58254.1
|
80-85℃
|
41.1%
|
Kang et al. 2011
|
Caldicellulosiruptor saccharolyticus
|
AAB06264.1
|
active at 85℃
|
38.1%
|
Albertson et al. 1997
|
3.2 Screening for mutation hotspots and generation of positive mutants
To identify the critical residues responsible for catalytic activity and stability of PulAR, we compared the protein sequences and structures of neutrophilic type I pullulanases PulA and PulAR with the acidophilic pullulanases, of which optimum conditions were at 50-60°C and pH 4.5-6.0. Thus, the sequences of pullulanase from Bapul and Bnpul were selected to align with the neutrophilic type I pullulanases (Figure 1a), and the difference in the amino acid residues among the above pullulanases within 8 Å of the catalytic triad was explored (Figure 1b). Finally, six mutants A365V, T399S, V401T, V401C, Y491V, and T504V, were generated. Besides, the single mutant H499A was also constructed.
3.3 Characterization Of Pular Mutants
First, the pullulanase activities of seven mutants were assayed at pH 5.0 and pH 6.0, respectively, and the activity ratio of WT and its mutants at pH 5.0 to that at pH 6.0 (ApH5.0/ApH6.0) were evaluated. As described in Table S2, ApH5.0/ApH6.0 of WT-PulAR and its mutants (A365V, T399S, V401T, V401C, Y491V, and T504V) were 0.20, 0.49, 0.12, 0, 0.75, 0.19, and 0.29 respectively. Therefore, we combined the positive mutations A365V, V401C, and T504V, and the mutation H499A, generating three combined mutants, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A.
We characterized the three combined mutants PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A, as well as two single mutants PulAR-A365V and PulAR-V401C. As shown in Figure 2, the optimum temperature (Topt) of PulAR was 55℃, and these of the mutants PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A were 55, 55, 60, 60, 60, and 65℃, respectively. Compared with WT, the Topt of the mutant PulAR-A365V-V401C-T504V-H499A was increased by 10℃. In addition, at 60℃ and pH 6.0, the specific activities of WT and its mutants PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-V401C-T504V-H499A were 24.4, 37.8, 43.3, 48.9, 68.9, and 87.8 U/mg. The optimum pH of PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, and PulAR-A365V-V401C-T504V was 6.0, which was similar to that of the WT. At 60℃ and pH 5.0, the specific activities of PulAR and its mutants PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A were 4.4, 10.0, 14.4, 32.2, and 40.0 U/mg, respectively. Among them, the specific activity of the quadruple mutant PulAR-A365V-V401C-T504V-H499A was 8.1-fold higher than that of WT.
To evaluate the thermostability, the enzymes were incubated at 60℃ and pH 6.0, and the residual activities were assayed after varying incubation times. As shown in Table 2, all the mutants PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A displayed enlarged half-lives. At 60 and 65 oC, the half-lives (t1/2) of PulAR were only 4.8 and 2.5 h, respectively, whereas those of the quadruple mutant were 17.5 and 10.3 h, which were 2.65 and 3.12-fold higher than those of PulAR. The stabilities of PulAR under the acidic conditions (pH 4.5 and 5.0) were also significantly enhanced. The half-lives of PulAR were 5.4 and 6.1 h at pH 4.5 and 5.0, respectively, whereas those of the quadruple mutant PulAR-A365V-V401C-T504V-H499A displayed longer half-lives of 13.9 and 17.3 h, respectively (Table 3). The structures of PulAR and its mutants were modeled to investigate the mechanisms of the enhanced thermostability and pH stability. A365 is buried in the internal of the protein. As shown in Figure 3, the mutation A365V introduces two extra hydrophobic interactions F432-V365 and F434-V365 while maintaining the two hydrogen bonds V365-R433 and D435-V365. For the mutation V401C, the residue Cys also forms a hydrogen bond with the residue T399 and a tighter hydrophobic interaction with H369 like the residue V401. Because of the mutation V401C is located on the protein surface, the stability might be enhanced by the hydrogen bonds formed between C401 and solvent water molecules. Similar to the mutation Y477A in our previous report, the solvent accessibility of the residue at the position 499 was reduced from 29.9 to 2.4, making the protein structure of PulAR more compact (Li et al. 2015). Replacing T504 with Val possessing an extra CH3 group reinforces the hydrophobic protein interior, leading to enhancement of the thermostability and pH stability(Tai et al. 2011).
Table 2
Half-lives of WT-PulAR and its variants at 60 and 65oC.
Mutant
|
60 oC
|
65 oC
|
kd (1/h)
|
t1/2 (h)
|
kd (1/h)
|
t1/2 (h)
|
WT-PulAR
|
0.14
|
4.8± 0.2
|
0.28
|
2.5± 0.1
|
PulAR-A365V
|
0.12
|
5.9± 0.5
|
0.17
|
4.1± 0.2
|
PulAR-V401C
|
0.12
|
6.0± 0.1
|
0.17
|
4.1± 0.1
|
PulAR-A365V-V401C
|
0.07
|
9.9± 0.2
|
0.10
|
6.7± 0.4
|
PulAR-A365V-V401C-T504V
|
0.05
|
13.2± 0.1
|
0.08
|
9.2± 0.2
|
PulAR-A365V-V401C-T504V-H499A
|
0.04
|
17.5± 0.1
|
0.07
|
10.3±0.3
|
Table 3
Half-lives of WT-PulAR and its variants at pH 4.5 and 5.0.
Mutant
|
pH4.5
|
pH5.0
|
kd (1/h)
|
t1/2 (h)
|
kd (1/h)
|
t1/2 (h)
|
WT-PulAR
|
0.13
|
5.4 ± 0.3
|
0.11
|
6.1 ± 0.5
|
PulAR-A365V
|
0.10
|
7.0 ± 0.4
|
0.08
|
8.5 ± 1.4
|
PulAR-V401C
|
0.10
|
7.1 ± 0.6
|
0.08
|
8.4 ± 0.3
|
PulAR-A365V-V401C
|
0.07
|
9.4 ± 0.3
|
0.06
|
10.8 ± 0.2
|
PulAR-A365V-V401C-T504V
|
0.06
|
11.1± 0.4
|
0.05
|
13.6± 1.0
|
PulAR-A365V-V401C-T504V-H499A
|
0.05
|
13.9 ± 1.2
|
0.04
|
17.3± 1.2
|
3.4 Kinetic Parameters Of Wt-pular And Its Mutants
WT-PulAR and its mutants were subjected to kinetic analysis at 60 ℃, pH 5.0 and 6.0, respectively. Compared with WT, at pH 6.0, 60 oC, the KM values of PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A decreased by 7.3%, 7.3%, 24.4%, 33.5%, and 53.0%, respectively, while the kcat values increased by 39.1%, 54.3%, 168.9%, 193.7%, and 254.3%, respectively (Table 4). In addition, at pH 5.0, 60 oC, the KM values of PulAR-A365V, PulAR-V401C, PulAR-A365V-V401C, PulAR-A365V-V401C-T504V, and PulAR-A365V-V401C-T504V-H499A decreased by 15.2%, 19.3%, 31.7%, 43.7%, 68.5%, respectively, while the kcat values increased by 37.7%, 45.7%, 154.8% 196.7%, and 230.7% (Table 5). Resultantly, the catalytic efficiencies (kcat/KM) of the quadruple mutant PulAR-A365V-V401C-T504V-H499A were 6.6 and 9.6-fold higher than those of PulAR, at pH 6.0 and 5.0, respectively. The catalytic efficiency of PulAR was enhanced by mutations identified by sequence alignment of the acidophilic pullulanase and neutrophilic pullulanase, and we further analyzed the roles of A365, V401, T504, and H499 in the structure-function relationship. The residues A365, V401, T504, and H499 lining the catalytic pocket are shown in Figure 1(b). They are located within 8 Å of the catalytic residues D435, E464, and D554. All the single mutations A365V, V401C, T504V, H499 and the superposition of mutations tend to confer increased flexibility of the active sites, therefore raising catalytic efficiency.
Table 4
Catalytic efficiencies of WT-PulAR and its mutants at 60 oC and pH 6.0.
Mutant
|
vmax (µmol min−1 mg−1)
|
KM (mg mL−1)
|
kcat (s−1)
|
kcat/KM (mL mg−1s−1)
|
WT-PulAR
|
31.6 ± 1.2
|
1.64 ± 0.20
|
52.7
|
32.1
|
PulAR-A365V
|
44.0 ± 2.3
|
1.52 ± 0.12
|
73.3
|
48.2
|
PulAR- V401C
|
48.8 ± 1.6
|
1.52 ± 0.50
|
81.3
|
54.2
|
PulAR-A365V-V401C
|
85.0 ± 2.5
|
1.24 ± 0.32
|
141.7
|
114.3
|
PulAR-A365V-V401C-T504V
|
92.9 ± 2.0
|
1.09 ± 0.11
|
154.8
|
142.0
|
PulAR-A365V-V401C-T504V-H499A
|
112.0 ± 3.1
|
0.77 ± 0.15
|
186.7
|
242.5
|
Table 5
Catalytic efficiencies of WT-PulAR and its mutants at 60 oC and pH 5.0.
Mutant
|
vmax (µmol min−1 mg−1)
|
KM (mg mL−1)
|
kcat (s−1)
|
kcat/KM (mL mg−1s−1)
|
WT-PulAR
|
25.6 ± 1.1
|
4.67 ± 0.12
|
42.7
|
9.1
|
PulAR-A365V
|
35.3 ± 0.3
|
3.96 ± 0.13
|
58.8
|
14.8
|
PulAR-V401C
|
37.3 ± 0.5
|
3.77 ± 0.20
|
62.2
|
16.8
|
PulAR-A365V-V401C
|
65.3 ± 1.2
|
3.19 ± 0.50
|
108.8
|
34.1
|
PulAR-A365V-V401C-T504V
|
76.0 ± 2.3
|
2.63 ± 0.28
|
126.7
|
48.2
|
PulAR-A365V-V401C-T504V-H499A
|
84.7 ± 1.6
|
1.47 ± 0.05
|
141.2
|
96.1
|
To further investigate the mechanism of the PulAR mutant against high temperature and acidic pH, MD simulation analysis of PulAR and the quadruple mutant AR-A365V-V401C-T504V-H499A was conducted. As shown in Figure 4, during the initial 6 ns for simulation, both the structures of PulAR and quadruple mutant AR-A365V-V401C-T504V-H499A are unstable. The entire protein conformation of the quadruple mutant became more stable than PulAR after 6 ns simulation time, which is consistent with stability enhancement under the thermophilic and acidic conditions.
In this work, we identified the active sites lining the catalytic pocket of PulAR by using a structure-guided consensus approach. SDM yielded four mutations A365V, V401C, T504V, and H499A, which showed beneficial effects on thermostability and acid resistance. In addition, the catalytic efficiencies of all the mutants were also significantly enhanced. Structural comparison indicated that increased internal hydrophobic interactions and the reduced solvent accessibility surface area are the main reasons for thermostability and acid resistance enhancement. In conclusion, it was proved that such a structure-guided consensus approach helps identify the critical residues for the improved catalytic performance of PulAR under thermophilic and acidic conditions, and the improved pullulanase exhibited great potential in the production of high-purity maltose syrup and other related starch processing industry.