Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

Abstract

Four chitinases were cloned and characterized from three strains isolated from a mudflat: Aeromonas sp. SK10, Aeromonas sp. SK15, and Chitinibacter sp. SK16. In SK10, three genes, Chi18A, Pro2K, and Chi19B, were found as a cluster. Chi18A and Chi19B were chitinases, and Pro2K was a metalloprotease. With combinatorial amplification of the genes and analysis of the hydrolysis patterns of substrates, Chi18A and Chi19B were found to be an endochitinase and exochitinase, respectively. Chi18A and Chi19B belonged to the glycosyl hydrolase family 18 (GH18) and GH19, with 869 and 659 amino acids, respectively. Chi18C from SK15 belonged to GH18 with 864 amino acids, and Chi18D from SK16 belonged to GH18 with 664 amino acids. These four chitinases had signal peptides and high molecular masses with one or two chitin-binding domains and, interestingly, preferred alkaline conditions. In the activity staining, their sizes were determined to be 96, 74, 95, and 73 kDa, respectively, corresponding to their expected sizes. Purified Chi18C and Chi18D after pET expression produced N,N'-diacetylchitobiose as the main product in hydrolyzing chitooligosaccharides and colloidal chitin. These results suggest that Chi18A, Chi18C, and Chi18D are endochitinases, that Chi19B is an exochitinase, and that these chitinases can be effectively used for hydrolyzing natural chitinous sources.

Keywords: Aeromonas sp.; Chitinibacter sp.; alkaline chitinases; chitooligosaccharides; endochitinases; exochitinase; gene cluster.

Figure 1

Growth and chitinolytic activities of the chitin-degrading microorganisms isolated from mudflats on LB containing 0.2% colloidal chitin (LBCC) agar plates. Each strain was streaked on LBCC agar plates and incubated at 30 °C for three days.

Figure 2

Phylogenetic tree of Aeromonas isolates SK10 and SK15 based on their 16S rRNA sequences. The phylogenetic tree was constructed using observed divergence method with bootstrap trials of 1000 in DNA/MAN.

Figure 3

Screening of chitinase-positive clones using LBCC/ampicillin plates. The clones were streaked on LBCC/ampicillin plates and incubated for three days at 30 °C. The positive clones were streaked on the same plates and compared with a control, which was transformed with a pUC19 vector.

Figure 4

Physical maps of the insert DNA of SK10-5 (A), and Chi18A and Chi19B (B).

Figure 4

Physical maps of the insert DNA of SK10-5 (A), and Chi18A and Chi19B (B).

Figure 5

Phylogenetic tree of the Chi18A and Chi19B proteins. The phylogenetic tree shows the evolutionary relatedness and levels of homology between the chitinolytic enzymes. The phylogenetic tree was constructed using the maximum likelihood method with bootstrap trials of 1000 in DNA/MAN.

Figure 6

Chitinase activities of subclones of three ORFs in clone SK10-5 on an LBCC plate. ORF1, ORF2, and ORF3 encoded Chi18A, Pro2K, and Chi19B, respectively.

Figure 7

Physical map of the insert DNA of SK15-36. The figure below of the physical map shows the result of BLASTp for Chi18C. The blue line and + symbol at the bottom show DNA of the clone and chitinase activity on the LBCC agar plates, respectively.

Figure 8

Phylogenetic tree of Chi18C. The phylogenetic tree was constructed using the maximum likelihood method with bootstrap trials of 1000 in DNA/MAN.

Figure 9

Physical map of the insert DNA of SK16-4366. The result of BLASTp for Chi18D is shown below the physical map. The blue line and + symbol at the bottom represent the DNA of the clone and chitinase activity on the LBCC agar plates, respectively.

Figure 10

Phylogenetic tree of the Chi18D protein. The phylogenetic tree was constructed using the maximum likelihood method with bootstrap trials of 1000 in DNA/MAN.

Figure 11

Effects of temperature (A), pH (B), thermostability (C), and metal ions (D) on the enzyme activity of Chi18A. The properties were characterized using the DNS method with colloidal chitin as a substrate.

Figure 12

Effects of temperature (A), pH (B), thermostability (C), and metal ions (D) on the enzyme activity of Chi19B. The properties were characterized using the DNS method with colloidal chitin as a substrate.

Figure 13

Effects of temperature (A), pH (B), thermostability (C), and metal ions (D) on the enzyme activity of Chi18C. The properties were characterized using the DNS method with colloidal chitin as a substrate.

Figure 14

Effects of temperature (A), pH (B), thermostability (C), and metal ions (D) on the enzyme activity of Chi18D. The properties were characterized using the DNS method with colloidal chitin as a substrate.

Figure 15

Confirmation of active sites of Chi18A, Chi18C, and Chi18D by site-directed mutagenesis. (A) Change of active site by site-directed mutagenesis from GAA and GAG (Glu) to GCA and GCG (Ala). (B) Chitinase activities of the mutants on an LBCC plate. (C) Chitinase activities of the crude extracts of the mutants.

Figure 16

Molecular masses of the chitinases produced by Chi18A, Chi19B, Chi18C, and Chi18D-containing clones. Lanes M, molecular weight standards; A, stained with Coomassie blue after SDS-PAGE; Lanes B, activity stained with MUCh₂.

Figure 17

SDS-PAGE (Lanes 1–4) and activity staining after renaturation treatment of chitinase (Lane 5) expressed in E. coli BL21 (DE3) harboring pET-28a(+)/chi18C (A) or/chi18D (B). Each panel represents: Lane 1, standard marker; Lane 2, the crude extract from each clone; Lane 3, the crude extract from each clone induced by IPTG; Lane 4, the purified chitinase using an Ni-NTA column from each clone; Lane 5, activity staining of the purified chitinase Chi18C or Chi18D from each clone.

Figure 18

Characterization of Chi18C (A–D) and Chi18D (E–H). Effects of temperature (A,E), pH (B,F), thermostability (C,G), and metal ions (D,H) on purified Chi18C and Chi18D activities. The properties were characterized using the DNS method with colloidal chitin as a substrate.

Figure 19

TLC profile of purified Chi18C (A,B) and Chi18D (C,D). TLC analysis of hydrolysis products of chitooligosaccharides (A,C) and colloidal chitin (B,D) as substrates for Chi18C and Chi18D. In (A,C): E₂ to E₆, N,N’-diacetylchitobiose (Ch₂) through to hexa-N-acetylchitohexaose (Ch₆) reacted with the enzyme. In (B,D): the reaction aliquots were sampled at the designated time above. The standards used were N-acetyl-D-glucosamine (Ch₁), N,N′-diacetylchitobiose (Ch₂), N,N′,N″-triacetylchitotriose (Ch₃), tetra-N-acetylchitotetraose (Ch₄), penta-N-acetylchitopentaose (Ch₅), and hexa-N-acetylchitohexaose (Ch₆).

Figure 20

The predicted model map and 3D structures of Chi18A and Chi18C. For Chi18A, the N-terminal part- (24–565) and C-terminal part- (155–815) containing structures were predicted with the model templates of Chitinase A (PDB code: 1 x 6l.1.A; identity of 74.42%) and Chitinase60 (PDB code: 4mb3.1.A; identity of 18.40%), respectively. For Chi18C, the N-terminal part- (155–812) and C-terminal part- (24–562) containing structures were predicted with Chitinase A (PDB code: 1ffr.1.A; identity of 76.02%) and Chitinase60 (PDB code: 4hme.1.B; identity of 17.62%), respectively, like Chi18A.

Figure 20

The predicted model map and 3D structures of Chi18A and Chi18C. For Chi18A, the N-terminal part- (24–565) and C-terminal part- (155–815) containing structures were predicted with the model templates of Chitinase A (PDB code: 1 x 6l.1.A; identity of 74.42%) and Chitinase60 (PDB code: 4mb3.1.A; identity of 18.40%), respectively. For Chi18C, the N-terminal part- (155–812) and C-terminal part- (24–562) containing structures were predicted with Chitinase A (PDB code: 1ffr.1.A; identity of 76.02%) and Chitinase60 (PDB code: 4hme.1.B; identity of 17.62%), respectively, like Chi18A.

Figure 21

The predicted model map and 3D structure of Chi19B. The model templates of the N-terminal part- (42–427) and C-terminal part- (481–592) containing structures were chitinase C (PDB code: 1wvv 2.B, identity of 32.95%) and cellulose-binding protein (PDB code: 2yhg 1.A, identity of 22.73%).

Figure 22

The predicted model map and 3D structure of Chi18D. The model templates of the N-terminal part- (31–161) and C-terminal part- (226–661) containing structures were deacetylase DA1 (PDB code: 4ny2.1.A; identity of 34.52%) and chitinase B (PDB code: 1kfw.1.A; identity of 44.01%), respectively.