A molecular study on recombinant pullulanase type I from Metabacillus indicus

Abstract

Despite the great potential of cold-adapted pullulanase type I in tremendous industrial applications, the majority of commercialized pullulnases type I are of mesophilic and thermophilic origin so far. Hence, the present study underlines cloning, heterologous expression in Escherichia coli, characterization, and in silico structural modeling of Metabacillus indicus open reading frame of cold-adapted pullulanase type I (Pull_Met: 2133 bp & 710 a.a) for the first time ever. The predicted Pull_Met tertiary structure by I-TASSER, was structurally similar to PDB 2E9B pullulanase of Bacillus subtilis. Purified to homogeneity Pull_Met showed specific activity (667.6 U/mg), fold purification (31.7), molecular mass (79.1 kDa), monomeric subunit and Km (2.63 mg/mL) on pullulan. Pull_Met had optimal pH (6.0) and temperature (40 ^oC). After 10 h pre-incubation at pH 2.6-6.0, Pull_Met maintained 47.12 ± 0.0-35.28 ± 1.64% of its activity. After 120 min pre-incubation at 30 ^oC, the retained activity was 51.11 ± 0.29%. At 10 mM Mn²⁺, Na²⁺, Ca²⁺, Mg²⁺, and Cu²⁺ after 30 min preincubation, retained activity was 155.89 ± 8.97, 134.71 ± 1.82, 97.64 ± 7.06, 92.25 ± 4.18, and 71.28 ± 1.10%, respectively. After 30 min pre-incubation with Tween-80, Tween-20, Triton X-100, and commercially laundry detergents at 0.1% (v/v), the retained activity was 141.15 ± 3.50, 145.45 ± 0.20, 118.12 ± 11.00, and 90%, respectively. Maltotriose was the only end product of pullulan hydrolysis. Synergistic action of CA-AM21 (α-amylase) and Pull_Met on starch liberated 16.51 g reducing sugars /g starch after 1 h at 40 ^oC. Present data (cold-adeptness, detergent stability, and ability to exhibit starch saccharification of Pull_Met) underpins it as a promising pullulanase type I for industrial exploitation.

Keywords: Biochemical characterization; Cold-adapted pullulanase type I; Heterologous expression; In silico structural modeling; Metabacillus indicus.

Fig. 1

 A) 12% SDS-PAGE showing purification steps of Pull_Met using Nickel -affinity agarose chromatography. Lane M: Protein ladder. Lane 1: purified Pull_Met after Nickel -affinity agarose chromatography column. Lane 2: crude cell lysate of induced recombinant E. coli BL21 (DE3) Rosetta cells. B) 10% native-PAGE showing the multimerization status of Pull_Met. Lane M: Protein ladder. Lanes (1–4): Pull_Met purified to homogeneity using nickel affinity chromatography column.

Fig. 2

pH-temperature profile of purified to homogeneity Pull_Met. A) Pull_Met activity profile over pH range 2.6–10.6. B) pH stability profile of Pull_Met over pH range 2.6–10.6. (C) Pull_Met activity profile over temperature range 5–60 °C. (D) Temperature stability profile of Pull_Met over temperature range 15–45 °C. Results are the mean of experimental readings performed in triplicate ± SE.

Fig. 3

 A) Stability profile of Pull_Met in presence of some detergents at three tested concentrations: 0.1, 0.25, and 0.5 mM. Control: without any pretreatment with detergents. Values are the average of three readings ± SE. B) Pull_Met stability in presence of solvents. Values are the average of three readings ± SE.

Fig. 4

TLC showing pullulan end products hydrolysis by Pull_Met. Panel A: G: glucose standard. S: undigested pullulan substrate. Panel B: G: glucose standard, M: maltose standard, P1: pullulan end products of hydrolysis (5 µL) and P2 pullulan end products of hydrolysis (10 µL): by Pull_Met. Panel C: G: glucose standard. St: standard end products of starch hydrolysis. Panel D: G: glucose standard, D: Dextrin end products of hydrolysis.

Fig. 5

Synergistic action of gradual concentrations of Pull_Met with 21.28 U/mg CA-AM21 on raw ex potato starch saccharification. Values are the average of three readings ± SE.

Fig. 6

Multiple sequence alignment between Pull_Met and other pullulanases type I from other species. A: the amino acid sequence of pullulanase type I signature motif highlighted by rectangle. B: the amino acid sequence of conserved region I in GH13 family highlighted by rectangle. C: the amino acid sequence of conserved region II in GH13 family highlighted by rectangle. D: the amino acid sequence of conserved region III in GH13 family highlighted by rectangle. E: the amino acid sequence of conserved region IV in GH13 family highlighted by rectangle. The accession number of pullulanases were AEW23439.1 (Anoxybacillus sp.), WP_004439017.1 (B. methanolicus), WP_251605681.1 (Priestia koreensis), UJT32179.1 (Priestia megaterium), UZX50359.1 (M. indicus), 2E8Y (AMyx protein of Bacillus cereus), WP_068759737 (Turicibacter sp.), WP_010899405.1 (Halakalibacterium halodurans), WP_011064811.1 (Oceanobacillus iheyensis), WP_008826932.1 (Haloplasma contractile), ACN58254.1 (Thermatoga neapolitana), WP_002277520.1 (Streptococcus mutans), WP_000283071.1 (Streptococcus pneumoniae), WP_067193616.1 (Strreptocccus sp.), and AVI10282.1 (Alkalihalophilus pseudofirmus). The catalytic triad residues: Asp, Glu, and Asp are indicated by asterisks

Fig. 7

 A) Predicted 3D structure of Pull_Met, generated by I-TASSER online program and visualized by PyMOL program, in a cartoon view showing α/β fold hydrolase using crystal structure PDB 2E9B of pullulanase type I from Bacillus subtilis str. 168. B) Cartoon view of predicted 3D structure of Pull_Met showing the catalytic triad residues at Asp ⁴¹⁰ (in blue spheres), Glu ⁴³⁹ (in yellowish orange spheres), and Asp ⁵²³ (in red spheres). C) Superposition, generated by TM-align Structural Alignment program and visualized by PyMOL program, between predicted 3D structure of Pull_Met (blue) and PDB hit 2e9b (green) in a cartoon view. D) Schematic representation of Pull_Met showing predicted domain organization as deduced c analysis of amino acid sequence on InterPro online program.