Abstract
Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.
Introduction
Chitin is a highly insoluble β-1, 4-liked polymer of N-acetylglucosamine (GlcNAc), and is the second most abundant polysaccharide (next only to cellulose) in nature. For the complete hydrolysis of chitin to GlcNAc, concerted action of chitinase (EC 3.2.1.14) and β-N-acetylglucosaminidase (EC 3.2.1.30) is essential. Chitin was extracted as two allomorphs, namely α- and β- forms [1]. The structures of α- and β forms of chitin differ only in the arrangement of piles of chains. Alternate chains are antiparallel in α-chitin, whereas they are all parallel in β-chitin. Among the chitin variants, α-chitin is the most abundant biopolymer in the nature. It occurs in fungal and yeast cell walls, krill, lobster and crab tendons and shells, and in shrimp shells, as well as in insect cuticle. Chitinase cleaves the glycosidic linkages between the adjacent GlcNAc residues to produce soluble oligosaccharides, which are further hydrolysed to GlcNAc by β-N-acetylglucosaminidases. Chitinase genes from bacteria have been cloned from both terrestrial and marine environments [2], [3]. Biochemical properties, catalytic mechanisms, and tertiary structures of chitinases were widely reported [4], [5]. A processive mechanism that improves substrate accessibility is generally considered favourable. But, it might in fact slow down enzymes. Improving substrate accessibility has been a key issue because this might reduce the need for using processive enzymes, which are intrinsically slow. Furthermore, carefully selected substrate-disrupting accessory proteins or domains might provide novel tools to improve substrate accessibility, and thus contribute to more efficient enzymatic processes [6].
Efficient chitin degradation also depends on the action of a family 33 chitin binding proteins (CBPs). The CBPs bind to the insoluble crystalline chitin, leading to structural changes and increased accessibility of substrate. The function of family 33 CBPs was first demonstrated for Sm CBP21 [7]. The details of CBPs and their binding preferences are given in Table 1. Studies of Sm CBP21 revealed that the protein has a “budded” fibronectin type 3-fold consisting of two β-sheets, arranged as a compact β-sheet sandwich-fold surface, having a at conserved region that binds chitin through interactions mediated mainly by polar amino acids [7], [8]. Conserved aromatic residues that have been suggested previously to play a role in chitin binding [9] were found in the interior of the protein, seemingly incapable of interacting with chitin. Sm CBP21 was designated as “chitin oxidohydrolase” as it acts on the surface of crystalline chitin, to introduce chain breaks and generates oxidized chain ends, promoting further degradation by chitinases [10]. Swapping of the chitin-binding domain in Bacillus chitinases improved the substrate binding affinity, and conformational stability [11].
Table 1. Details of bacterial CBPs and their binding preferences.
| CBP Name | Source | Binding substrates | References |
| CBP21 | S. marcescens | High preference to β-chitin followed by regenerated chitin and colloidal chitin | [15] |
| ChbB | B. amyloliquefaciens | Binds to both α- and β-chitin but shows preference to β-chitin | [17] |
| LlCBP33A | Lactococcus lactis | Equally well to α- and β-chitin, followed by Avicel, colloidal chitin and chitin beads | [4] |
| CHB1 | St. olivaceoviridis | Strictly to α- chitin | [25] |
| CHB2 | St. reticuli | Strictly to α- chitin | [26] |
| CHB3 | St. coelicolor | Most preferably to α -chitin followed by β -chitin | [27] |
| CbpD | P. aeruginosa | Colloidal chitina | [28] |
| E7 | Thermobifida fusca | Equally well to α- and β-chitin followed by bacterial microcrystalline cellulose | [29] |
| E8 | Thermobifida fusca | Preferentially to β-chitin followed by α-chitin and microcrystalline cellulose | [29] |
| Cbp50 | B. thuringiensis | Preferentially to β-chitin followed by α-chitin, colloidal chitin and cellulose | [18] |
| EfCBM33A | E. faecalis | Binds both α- and β-chitin, but slightly more protein binds to β-chitin | [19] |
Details of binding to other substrates not available.
S. proteamaculans 568, a member of family Enterobacteriaceae, was isolated as a root endophyte from Populus trichocarpa [12]. According to the Carbohydrate Active enZyme data base (CAZy-http://www.cazy.org/) [13] S. proteamaculans 568 has at least eight genes involved in chitin turnover, coding for four family 18 chitinases (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD), three family 33 CBPs (Sp CBP21, Sp CBP28 and Sp CBP50), and a family 20 N-acetylhexosaminidase (Sp CHB). The present study, describes the cloning and characterization of three CBPs from S. proteamaculans 568 and their synergy with Sp chitinases in degradation of natural chitin variants.
Results
Amplification and cloning of CBPs from S. proteamaculans 568
Three Sp cbp genes were amplified using gene specific primers with gDNA of S. proteamaculans 568 as template. The three Sp CBPs were predicted to contain N-terminal leader peptide directing sec-dependent secretion. Signal peptide was predicted using the SignalP server (http://www.cbs.dtu.dk/services/SignalP/). The genes were cloned without the signal peptide-encoding portion (Sp cbp21: 81 bp, Sp cbp28: 66 bp and Sp cbp50: 63 bp). The amplicons 0.51 kb of Sp cbp21, 0.76 kb of Sp cbp28 and 1.35 kb of Sp cbp50 were cloned in the Nco I and Xho I sites of pET 22b (+) and Eco RI and Xho I sites of pET- 28a (+), respectively.
The three Sp CBPs were over expressed with a C-terminal His-tag in E. coli. The expressed Sp CBPs were separated either from periplasmic fraction (Sp CBP21 and Sp CBP28) or from whole cell lysate (Sp CBP50) as soluble proteins, and purified using Ni-NTA agarose chromatography. The PelB signal sequence in pET-22 b (+) directs the expressed Sp CBP21 and Sp CBP28 proteins towards periplasmic space. SDS-PAGE analysis of the purified Sp CBPs revealed approximate molecular weight of 18.6, 28.0 and 50.0 kDa, which correspond to Sp CBP21, Sp CBP28 and Sp CBP50, respectively (Figure S1).
Substrate binding preference of Sp CBPs
The binding preference of Sp CBPs was assessed by incubating the protein with different insoluble polymeric substrates α-chitin, β-chitin and colloidal chitin and Avicel. The amount of Sp CBP bound to the respective substrate was analyzed by determining the protein concentration in the supernatant of the reaction mixture after 24 h of incubation. Sp CBP28 did not bind to any of the test substrates (data not shown). Both Sp CBP21 and Sp CBP50 bound equally high to β-chitin (86.2% and 77.0%), followed by colloidal chitin (68.8% and 65.6%), α-chitin (30.9% and 25.6%) and Avicel (25.9% and 19.3%) (Figures 1A and B).
Figure 1. Binding of Sp CBPs to insoluble polymeric substrates.
The reaction mixture (1 mL) containing 100 µg of Sp CBP21/Sp CBP50 and 1 mg of one of the insoluble substrates (α-chitin, β-chitin, colloidal chitin and Avicel) was incubated in 50 mM sodium phosphate buffer pH 7.0 under constant shaking at 1300 rpm at 37°C for 24 h. (A and B) The amount of bound protein was calculated as the difference in protein concentration before and after incubation with the insoluble substrates, (C and D) Decrease in free protein concentration after binding to α- and β-chitin was determined at different time points till 24 h. (A and C) Sp CBP21, (B and D) Sp CBP50. Vertical bars represent standard deviation of triplicate experiments.
The time course of binding was monitored for both the Sp CBPs as a function of time to find the time required for the Sp CBPs to get saturated with natural chitin variants. After separating the protein bound to chitin, the decrease in concentration of the unbound protein (remaining in the supernatant) was monitored at different time points up to 24 h. The binding of Sp CBP21 to β-chitin occurred rapidly and reached equilibrium within 6 h, while Sp CBP50 reached equilibrium by 12 h. On the other hand, Sp CBP21 and Sp CBP50 have established binding equilibrium to α-chitin by 12 h (Figures 1C and D).
Adsorption isotherms of Sp CBP21 and Sp CBP50 towards α- and β-chitin were estimated and plotted with fixed concentration of substrate and varied concentrations of the CBPs. The dissociation binding constants (K d) of α- and β-chitin were estimated from the non-linear regression function. The K d value of the Sp CBP21 to α-chitin (5.31±1.03 µM ) was much lower than the K d value of Sp CBP50 to α-chitin (9.34±1.67 µM ), whereas the K d value of Sp CBP21 to β-chitin (2.22±0.45 µM) was slightly lower than the K d value of Sp CBP50 to β-chitin (2.37±0.5 µM) (Figure 2). Binding of Sp CBPs to the soluble substrates was also examined especially to investigate whether Sp CBP28 binds at least to soluble substrates. Electrophoretic mobility of Sp CBPs did not change in presence or absence of glycol chitin, CM cellulose and laminarin substrates (Figure S2).
Figure 2. Equilibrium adsorption isotherms of Sp CBP21 and Sp CBP50 to α- and β-chitin.
The reaction assay (1 mL) containing 1.0 mg substrates (α- and β- chitin) and varied concentrations of Sp CBP21 and Sp CBP50 starting from 0 to 10.0 µM was incubated (Sp CBP21 with α- and β-chitin, 12 h and 6 h, respectively; Sp CBP50 with α- and β-chitin, 12 h) at 37°C. The reaction mixtures were centrifuged and concentration of bound protein (Pbound) and un-bound free protein (Pfree) was determined and plotted to fit into GraphPad Prism software version 5.0. All data sets were fitted to the equation for one-site binding by non-linear regression function, and to calculate B max and K d using GraphPad Prism software version 5.0. (A and B) The K d and B max values of Sp CBP21 and Sp CBP50 were shown in the inset table.
Homology modeling of Sp CBP21and Sp CBP50
Sequence alignment of Sp CBP21, Sp CBP28 and Sp CBP50 with Sm CBP21 displayed 93%, 55% and 18% identity, respectively. The surface-located polar residues of Sm CBP21 [7], [8] were highly conserved in Sp CBP21 (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) and Sp CBP50 (Tyr-48, Glu-49, Glu-54, His-108, Asp-176, and Asn-179). Sp CBP28 had only one (Asp-176) matching residue (Figure 3A), out of six conserved polar residues. Both Sp CBP21 and Sp CBP50, showed similar β-chitin binding preference as shown by Sm CBP21, because of sequence homology and presence of conserved polar surface residues.
Figure 3. Sequence alignment and domain organisation for Sp CBPs.
(A) Full-length sequences of Sp CBP21, Sp CBP28, Sp CBP50 and Sm CBP21 (CBP21 from S. marcescens) were aligned using clustalw2. Residues that are thought to be located in the binding surface for chitin present in Sm CBP21, Sp CBP21, Sp CBP50 and not present in Sp CBP28 are shaded in yellow (as derived from the crystal structure of Sm CBP21, as well as mutagenesis studies [7], [8]). Residue involved in the chitin-binding and functional properties of Sm CBP21 but also conserved in Sp CBP28 are shaded grey. The arrow indicates the terminal amino acid of the N-terminal signal sequence for respective CBPs. (B) The sequences of Sp CBPs were submitted to SMART domain data base (http://smart.embl-heidelberg.de/). The part indicated in red colour shows the signal peptide and the region Chitin_bind_3 indicates the chitin binding domain.
Sequence alignment and the SMART domain search data base revealed that the Sp CBP50 has N-terminal ChBD domain consisting of only 192 amino acids and the large part of the remaining sequence did not give any functional domain (Figure 3B). Sp CBP21 displayed high sequence identity to the sequences of the Sm CBP21 (93%). Sp CBP50 displayed only 55% of identity to Sm CBP21. The 3D structure models of Sp CBP21 and Sp CBP50 were generated using the template structure of Sm CBP21 (PDM ID: 2BEM) (Figures 4A and B). We have modeled only particularly ChBD region of the Sp CBP50 protein. The superimposition Cα atoms of the final model, on the template structure, gave a root mean square deviation (RMSD) of 0.182 Å and 0.164 Å for Sp CBP21 and Sp CBP50. Closer inspection of these structures revealed that the conformations of the several regions (α5, β4, β5, β6 and β7) were differing from the template structure (Figures 4C and D). Superimposition of Sp CBP21 and Sp CBP50 with the template revealed that the difference was mostly in the β-sheets.
Figure 4. The 3D models of Sp CBP21 and Sp CBP50.
(A and B) The models Sp CBP21 and ChBD region of Sp CBP50 were generated by Modeller9v8 (http://www.salilab.org/modeller/) using Sm CBP21 (PDB ID: 2BEM) as structure template. Residues important for chitin binding were shown in sticks representation with carbon, oxygen and nitrogen atoms colored light green, red and dark blue, respectively. The figures were prepared using PyMOL (http://www.pymol.org/), (C and D) Stereo view of the superimposed structure of Sp CBP21 and Sp CBP50 (green) with Sm CBP21 (red), respectively.
Synergism between Sp chitinases and Sp CBPs (Sp CBP21 & Sp CBP50) during degradation of α- and β-chitin
The hydrolytic efficiency of four Sp chitinases (Sp ChiA, Sp ChiB, Sp ChiC, and Sp ChiD) on natural chitin variants was estimated in the presence/absence of Sp CBP21/Sp CBP50 and/or an external electron donor (reduced glutathione). The results indicate that, upon addition of Sp CBP21/Sp CBP50, efficiency of hydrolysis of α- and β-chitin by all the four Sp chitinases increased, especially in the presence of a reduced glutathione. Combination of both enzymes and the external electron donor led to total solubilization of β-chitin within 24 h, whereas only small fraction of the α-chitin was solubilised under the same conditions. The reactions shown in Figure 5 were sampled for up to one week following the 24-h time point. All reactions containing β-chitin reached complete solubilization after about one week of incubation, whereas none of the samples containing α-chitin were degraded completely after one week (results not shown). All three Sp CBPs showed optimum binding at 35°C-40°C, while all four Sp chitinases were also optimally active at the same temperature range. So, the synergistic experiments were carried out at 37°C (data not shown).
Figure 5. Degradation of α- and β-chitin by Sp chitinases in the absence or presence of Sp CBP21, Sp CBP50 and reduced glutathione.
Reaction mixture (1 mL) containing 0.25 mg/mL of chitin substrates (α- and β-chitin), 1 µM of Sp chitinase (Sp ChiA/Sp ChiB/Sp ChiC/Sp ChiD) were incubated with 0.3 µM Sp CBP21 or Sp CBP50 and 1.0 mM reduced glutathione in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 7 days at 1000 rpm, after every 24 h, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments. (A and B) Degradation of α- and β-chitin by Sp chitinases in the presence/absence of Sp CBP21 and reduced glutathione (RG), (C and D) degradation of α- and β-chitin by Sp chitinases in the presence/absence of Sp CBP50 and reduced glutathione (RG). Sp CBP21+RG or Sp CBP50+RG: Sp CBP21/Sp CBP50 and reduced glutathione, Sp CBP21+ or Sp CBP50+: only Sp CBP21/Sp CBP50 without reduced gluthathione, Sp CBP21- or Sp CBP50 -: without Sp CBP21/Sp CBP50 and reduced glutathione.
Figure 5 shows that the synergism exhibited by Sp CBP50 with Sp chitinases was lower when compared to the Sp CBP21 in degrading natural chitin variants. This was almost compensated when reduced glutathione was supplemented to the Sp CBP50. The product formation efficiency by Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD increased by 0.38, 1.04, 0.99 and 5.75-fold, respectively on α-chitin in presence of Sp CBP21 and reduced glutathione (Figure 5A). On the β-chitin substrate, the products formation increased by 0.44, 3.28, 1.20, and 7.50- folds in the presence of Sp CBP21 and reduced glutathione (Figure 5B). In the presence of Sp CBP50, Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD efficiency of α-chitin hydrolysis increased by 0.35, 0.88, 0.72 and 5.11-fold, respectively (Figure 5C) where as it was 0.42, 3.12, 0.76 and 7.43-fold higher, respectively on β-chitin (Figure 5D). The increased product formation in presence of Sp CBP21 and Sp CBP50 was relatively more with Sp ChiD on α-chitin compared to other Sp chitinases, while on β-chitin, Sp ChiD and Sp ChiB were having major advantage with both the Sp CBPs. Dose-response studies of the effect of Sp CBP21 and Sp CBP50 on Sp ChiD efficiency showed that Sp ChiD displayed maximum degradation rates at both the Sp CBP's concentrations ≥0.2 µM (Figure S3). The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture of varied concentrations of Sp ChiD did not significantly increase the activity. Sp CBP21 appears to be compensating Sp CBP50 activity (Figure 6). Sampling of the synergistic mixtures (as above) at regular intervals, up to 24 h, also did not resulted improved product formation when compared to one time point sampling at 24 h (data not shown).
Figure 6. β-chitin hydrolysis enhancing effects of Sp CBP21 and Sp CBP50 with Sp ChiD.
Reaction mixture (1 mL) containing 0.25 mg/mL of β-chitin, 0.25 µM/0.50 µM/0.75 µM/1.0 µM Sp ChiD incubated individually with 0.3 µM of Sp CBP21/Sp CBP50 or combining both Sp CBP21 and Sp CBP50 (0.15 µM +0.15 µM/0.30 µM +0.30 µM), in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments.
Discussion
The ChBMs (chitin binding modules) are known to occur as discrete domains in chitinases and also exist independently as CBPs grouped in families 14, 18, and 33. Families 14 and 18 constitute small anti-fungal proteins that share a structurally similar chitin-binding motif [14]. Family 33 CBPs are mainly found in bacteria and viruses. Bacterial family 33 CBPs are expressed and secreted during chitin degradation. Sm CBP21 invades the chitin matrix to dissolve individual polymers, and make them more accessible to degradation by chitinases [8].
Analysis of genome sequence of S. proteamaculans 568 revealed the presence of genes coding for three CBPs of family 33. The Sp CBP21 was designated according to its homology to the reported Sm CBP21. Sp CBP21 showed high sequence identity to Sm CBP21 (93%). There were no reports on the presence of additional CBPs in S. marcescens. The two additional CBPs of S. proteamaculans were designated as Sp CBP28 and Sp CBP50 according to the estimated molecular weights (without the signal peptide) of these two proteins. S. proteamaculans 568 codes for at least 3 CBPs, while others produce either one or two CBPs (Table 1). The three Sp CBPs were arranged distantly in the genome of S. proteamaculans 568 and shares conserve 8 bp regions (5′-C(C/T/A) C(C/T) (T/G) G (C/A) (C/G)-3′) in the upstream sequences with other Sp chitinolytic genes (data not shown). Therefore, these Sp cbp genes might be co-ordinately controlled by the same regulatory protein(s) along with other Sp chitinolytic genes. Since S. proteamaculans 568 produced additional CBPs, characterization of CBPs in terms of their binding properties as well as synergism with Sp chitinases in chitin degradation was investigated.
The amino acid sequence of Sp CBP21 was BLAST at NCBI database to search for homologs. The result displayed 93% identity to Sm CBP21 (BAA31569), 57% to CBP from Bacillus cereus G9241 (EAL13960) and CBP from B. thuringiensis serovar tochigiensis BGSC 4Y1, 44% to Cbp21 from from B. anthracis str. CDC 684 (ACP12567), 41% to CHB2 from Streptomyces reticuli (EEM22267), 30% to CbpD from Stenotrophomonas sp. SKA14 (EED38588) and 27% to CbpD from Pseudomonas aeruginosa (AF196565). Sp CBP21 contained a signal peptidase site between amino acid residues Ala-27 and His-28. ChBD (Chitin binding domain) was present from His-28 through Asn-194.
Sp CBP28 showed 36% identity to CBP1 from Pseudoalteromonas piscicida (BAB79619) and CBP from Vibrio cholerae NCTC 8457 (EAZ71564), 33% to CBP from B. thuringiensis BMB171 (ADH07314) and CBP from B. cereus ATCC 1457 (AAP09751), and 31% to CBP from Streptomyces sp. e14 (EFF90245). A signal peptidase site was located between amino acid residues Ala-22 and Gln-23 of Sp CBP28. ChBD was present from His-39 through Asn-273.
The BLAST search for Sp CBP50 homologs displayed 62% identity to N-acetylglucosamine-binding protein A from Enterobacter cloacae subsp. cloacae ATCC 13047 (ADF60226), 57% to CBP21 from S. marcescens (BAA31569), 46% to CBP from Shewanella sp. HN-41 (ABK37291), 45% to CBP from Aeromonas hydrophila subsp. hydrophila ATCC 7966 (ABK37291), 40% to CHB2 from Streptomyces reticuli (CAA74695), and 24% to CbpD from Vibrio harveyi HY01 (EDL70242). Sp CBP50 contained a signal peptidase site between amino acid residues Ala-21 and His-22 and ChBD present from His-22 through Asp-188.
Binding studies of Sp CBPs to chitin variants and cellulosic substrates revealed that Sp CBP28 did not to bind to the test substrates. Sp CBP21 and Sp CBP50 were similar to the Sm CBP21, with maximum binding to β-chitin followed by α-chitin, colloidal chitin and Avicel (Figures 1A and B). As α-chitin has strong intersheet and intrasheet hydrogen bonding, compared to weak hydrogen bonding in intrasheets of β-chitin, the Sp CBP21 and Sp CBP50 preferably bound to β-chitin.
The difference in substrate preference was mainly attributed to the difference in the amino acid sequence of respective CBPs. The only available three-dimensional structure of close to Sp CBPs was Sm CBP21, which binds exclusively to β-chitin [15]. The combination of sequence and structural information with the results of site-directed mutagenesis showed that the surface of family 33 CBPs contains a patch of highly conserved, mostly polar residues (Tyr54, Glu55, Glu60, His114, Asp182, and Asn185), important for binding to chitin, and also for a positive effect on the efficiency of chitinase [8]. LlCBP33A which binds equally to α- and β-chitin had two substitutions in the conserved surface patch. Both these residues were known to be important for Sm CBP21 functionality [8]. Ser63 occurs at a position at which Sm CBP21 had a tyrosine (Tyr54), and other family 33 CBPs have tryptophan e.g. Trp57 in CHB1 from St. olivaceoviridis, which has been shown to be important for the ability of CHB1 to bind α-chitin [16]. Asn64 occurs instead of a Glu55 of Sm CBP21. The closest homologue of LlCBP33A is ChbB from B. amyloliquefaciens (66% sequence identity), which binds both α- and β-chitin [17]. ChbB differs from Sm CBP21 in the same two positions as LlCBP33A: Tyr54 is replaced by Asp62, and Glu55 is replaced by Asn63.
Alignment of the amino acid sequence of Sp CBPs with Sm CBP21revealed that all the amino acid residues that are important in chitin binding [7], [8] are conserved in Sp CBP21 (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) and Sp CBP50 (Tyr-48, Glu-49, Glu-54, His-108, Asp-176, and Asn-179), while Sp CBP28 showed only one conserved residue (Asp-176) (Figure 3A). Minimum homology and absence of important polar residues could be the reason for the inability of Sp CBP28 to bind to substrates. It remains to be confirmed whether Sp CBP28 has a role other than chitin binding. The presence or absence of conserved amino acid residues in CBPs, therefore, conferred substrate binding preference.
The binding of Sp CBP21 and Sp CBP50 to β-chitin occurred rapidly and reached equilibrium within 6 and 12 h, respectively. Sm CBP21 established binding equilibrium after 16 h of incubation [7]. Sp CBP21 and Sp CBP50 showed relatively slow binding to α-chitin and reached equilibrium by 12 h. LlCBP33A from Lactococcus lactis subsp. lactis established binding equilibrium by approximately 24 h of incubation with both α- and β-chitin [4]. In agreement with the binding assay, the lower K d values of Sp CBP21 and Sp CBP50 indicate that both these CBPs have high binding strength towards the β-chitin in comparison with α-chitin. Sp CBP21 and Sp CBP50, K d values towards the β-chitin were relatively higher, while B max values were lower when compared to the K d and B max values of reported CBPs from S. marcescens and B. thuringiensis serovar konkukian [7], [18]. None of the Sp CBPs bound to soluble substrates, as observed for Sm CBP21 by Vaaje-Kolstad et al., [7].
Sm CBP21 catalyzes cleavage of glycosidic bonds in crystalline chitin [10], opening up the inaccessible polysaccharide material for hydrolysis by normal glycoside hydrolases. Such unique enzymatic activity was discovered after detection of traces of previously unidentified chitooligosaccharides up on incubation of β-chitin nano whiskers with Sm CBP21. Vaaje-Kolstad et al., [7] reported that the CBPs bind to the insoluble crystalline substrate, leading to both structural changes and increased substrate accessibility to the Sm chitinases (Sm ChiA, Sm ChiB and Sm ChiC). The Sm CBP21 strongly promoted hydrolysis of crystalline β-chitin by Sm ChiA and Sm ChiC, while Sm ChiB it was essential for complete degradation, and Sm CBP21 activity was boosted by external electron donor [10]. Vaaje-Kolstad et al., [4], [19] also showed that the LlCBP33A and EfCBM33A increased the hydrolytic efficiency of LlChi18A and EfChi18A, respectively to both α- and β-chitin. These results show the general importance of CBPs in chitin turnover.
Among the four Sp chitinases, Sp ChiA, Sp ChiB and Sp ChiC released chitobiose as major end product [20], while Sp ChiD released GlcNAc from chitin substrates (based on HPLC) (data not shown). Hydrolysis of natural chitin variants by four Sp chitinases in the presence of Sp CBP21 and Sp CBP50 showed that efficiency of all the four Sp chitinases increased with both α- and β-chitin (Figure 5). The Sp chitinases were less active on α-chitin (data not shown) and Sp CBP21 and Sp CBP50 had only minor binding preference to α- chitin. Therefore, there was no significant increase in the product formation on α-chitin. The addition of Sp CBP21 and Sp CBP50 had only minor effect on hydrolysis of β-chitin by Sp ChiA and Sp ChiC, while efficiency of Sp ChiB and Sp ChiD increased significantly high. These results are in line with the earlier report on Sm ChiB [8] that was dependant on Sm CBP21 in β-chitin degradation. Interestingly, in both the organisms, the cbp21 gene is located 1.5 kb downstream to the Sm chiB. It was even reported that Sm CBP21 was produced along with three chitinases, Sm ChiA, Sm ChiB and Sm ChiC [15]. Overall, with both the chitin substrates (α- and β-chitin) Sp ChiD obtained major benefit from Sp CBP21 and Sp CBP50. Sp ChiD is a single GH18 domain enzyme that exhibited lower activity on both α- and β-chitin when compared to other Sp chitinases. CBP-mediated enhancement of substrate availability increased the efficiency of Sp ChiD. Sp CBP21 and Sp CBP50 are useful to convert the chitin biomass into production of chitooligosaccharides, which are useful in agriculture, food, and pharmaceutical industries. The uniqueness of Sp CBP28 is being investigated in terms of its importance in biology of S. proteamaculans 568.
Materials and Methods
Chemicals and enzymes
Restriction enzymes, T4 DNA ligase, and Pfu DNA polymerase were from MBI Fermentas (Ontario, Canada). Primers were procured from Eurofins India (Bangalore, India). Isopropyl-β-D-thiogalactoside (IPTG), ampicillin, kanamycin, chloramphenicol and all other chemicals were purchased either from Sigma–Aldrich (Missouri, USA), or Merck (Darmstadt, Germany), or Hi-media labs (Mumbai, India). The polymeric substrates α- and β-chitin were kindly provided by Mahtani Chitosan (Veraval, India). Colloidal chitin (CC) was prepared according to Berger and Reynolds [21].
Bacterial strains, plasmids, and media
Serratia proteamaculans 568 was grown in Luria-Bertani (LB) broth at 28°C for 16 h for the extraction of gDNA (QIAgen, Duesseldorf, Germany). Plasmid vectors pET 22b(+), pET 28a(+), and Escherichia coli Rosetta-gami 2(DE3) were used as vector and host (Novagen, Darmstadt, Germany) for expression, respectively. To express CBP genes, E. coli Rosetta-gami 2(DE3) carrying pET 22b(+) or pET 28a(+) was grown in LB broth with ampicillin (100 µg/mL) and chloramphenicol (25 µg/mL) or kanamycin (50 µg/mL) and chloramphenicol (25 µg/mL), respectively.
Amplification and cloning of Sp CBPs
Three genes encoding CBPs (Sp cbp21, Sp cbp28 and Sp cbp50; GenBank accession no. ABV42576.1, ABV42205.1, and ABV43333.1, respectively) were amplified from the gDNA by referring to the annotated sequence of S. proteamaculans 568 at 55°C annealing temperature using gene specific forward and reverse primers listed in Table 2. Expression vectors, and the amplicons were separately digested with Nco I and Xho I (pET 22b(+), Sp cbp21 and Sp cbp28), and Eco RI and Xho I (pET 28a(+) and Sp cbp50), gel purified and ligated using T4 DNA ligase at 16°C for 16 h. The resultant plasmids were designated as pET 22b–Sp cbp21, pET 22b–Sp cbp28 and pET 28a–Sp cbp50 to express Sp CBP21, Sp CBP28 and Sp CBP50, respectively in E. coli.
Table 2. List of primers used for the amplification of genes coding for chitin binding proteins and chitinases from Serratia proteamaculans 568.
| Gene I.D | Gene | Primer | Primer sequencesa (5′<$>\vskip -1\scale 40%\raster="rg1"<$>3′) | Restriction site |
| ABV42576.1 | Sp cbp21 | Forward | AAT AAC CAT GGT TCA CGG CTA TGT CGA AAC | Nco I |
| Reverse | GCA TAC TCG AGT TTA GTC AAA TTA ACG TC | Xho I | ||
| ABV42205.1 | Sp cbp28 | Forward | ATA CAC CAT GGT TCA GGA GCA AGT TTC CAC TAC | Nco I |
| Reverse | AAT CAC TCG AGG CCT TTG ATA TTG ACG TCA C | Xho I | ||
| ABV43333.1 | Sp cbp50 | Forward | TCA AGA ATT CCA TGG TTA TGT TGA ATC GCC G | Eco RI |
| Reverse | ATA AAC TCG AGT CAG TTT TTT AAT ATC CAG GCT TGT TGC C | Xho I | ||
| ABV39247.1 | Sp chiA | Forward | CAA TAA CCA TGG CCG TAC CGG GTA AGC CTA C | Nco I |
| Reverse | AAT AAC TCG AGT TGC GTG CCG GCG CTG TTG | Xho I | ||
| ABV40327.1 | Sp chiB | Forward | CAT AAC CAT GGT GTC CGA ACG TAA AGC CGT TAT TG | Nco I |
| Reverse | AAT AAC TCG AGT GCC ACG CGG CCC ACT TTC | Xho I | ||
| ABV42574.1 | Sp chiC | Forward | GCT CAC CAT GGT GAG CAC CAA TAA TAT TAT CAA TGC | Nco I |
| Reverse | AAT AAC TCG AGG GCG GTC AAC TGC CAC AG | Xho I | ||
| ABV41826.1 | Sp chiD | Forward | TAA TAC CAT GGG TGC CGG CAT GGC TCA TG | Nco I |
| Reverse | AAT AAC TCG AGC TGT TTC CCG TTA ATC C | Xho I |
Sequences underlined represent restriction sites.
Expression and purification of Sp CBPs
Expression and purification of Sp CBP21, Sp CBP50 and Sp CBP28 were done as described by Neeraja et al., [22], except that the Sp CBP50 was isolated from whole cell lysate by sonicating the cell pellet. The cell pellet was suspended in Ni-NTA equilibration buffer (50 mM NaH2PO4, 100 mM NaCl and 10 mM imidazole pH 8.0). The cells were lysed by sonication at 20% amplitude with 30×15 s pulses (with 20 s delay between pulses) on ice, with a Vibra cell Ultrasonic Processor, converter model CV33, equipped with a 3 mm probe (Sonics, Newtown, CT, USA). To pellet the insoluble cell debris, sonicate was centrifuged at 15,200× g for 10 min at 4°C. The expressed protein was purified using Ni-NTA column as the expressed protein having C-terminal His-tag. After purification, the Sp CBPs were buffer exchanged with 50 mM sodium phosphate buffer pH 7.0 using Macrosep Centrifugal Devices (Pall Corporation, USA), and stored at 4°C until use.
Protein measurement
Purified Sp CBPs were quantified by BCA (bicinchonic acid) protein assay kit (Novagen, USA) using a standard calibration curve constructed from BSA (bovine serum albumin). For the chitin binding assay, protein concentration was measured from the absorption at 280 nm using the molar extinction coefficients (ε) calculated from the amino acid composition of the protein as described by Pace et al., [23].
Insoluble substrate binding specificity
Insoluble substrate binding of Sp CBPs was done as described by Vaaje-Kolstad et al., [7] with slight modifications. The substrates, α-chitin, β-chitin, colloidal chitin and Avicel were used as insoluble substrates, and BSA was used as a background control for nonspecific adsorption. The binding mixture (1 mL) was incubated for 24 h at 37°C with vigorous shaking at 1300 rpm on thermomixer (Thermomixer comfort; Eppendorf, Hamburg, Germany).
Time course binding of Sp CBP21 and Sp CBP50 towards α- and β-chitin
To study the time at which the binding of Sp CBP21 and Sp CBP50 (described above) was getting saturated with natural chitin variants (α- and β-chitin) was assessed at different time points up to 24 h.
Adsorption isotherms of Sp CBP21 and Sp CBP50 towards α- and β-chitin
Adsorption isotherms of Sp CBP21 and Sp CBP50, towards α- and β-chitin, were obtained as described by Vaaje-Kolstad et al., [7] with minor modification. Varied concentration of an Sp CBP, up to 10.0 µM, was incubated with α-/β-chitin for different saturation periods: 12 h for α-chitin and 6 h for β-chitin with Sp CBP21, and 12 h for both α- and β-chitin with Sp CBP50.
Soluble substrate binding specificity
Binding of Sp CBPs to soluble polysaccharides (glycol chitin, laminarin and CM cellulose) was evaluated by affinity electrophoresis as described by Hardt and Laine [24] with slight modifications. Proteins (10 µg of Sp CBPs and non-interacting BSA) were electrophoresed in 8.0% polyacrylamide gels impregnated with substrates (glycol chitin or laminarin or CM-cellulose) under non-denaturing conditions at 4°C. The gels were visualized by staining with Coomassie blue G-250.
Sequence alignment for Sp CBPs and homology modelling of Sp CBP21and Sp CBP50
All three Sp CBPs from S. proteamaculans were aligned with Sm CBP21 using clustalw2 (www.ebi.ac.uk/Tools/msa/clustalw2/). A 3D structure models of Sp CBP21 and Sp CBP50 were generated using the template structure of Sm CBP21 (PDM ID: 2BEM) by Modeller9v8 (http://www.salilab.org/modeller/). About 40 models and corresponding Ramachandran plots were generated for each protein to check the protein structure quality using PROCHECK. The figures were prepared using PyMOL (http://www.pymol.org/).
Synergistic effect of Sp CBP21 and Sp CBP50 with Sp chitinases in chitin degradation.
Chitin degradation assay was performed as described by Vaaje-Kolstad et al., [19] with few modifications. A standard 1 mL reaction mixture containing 0.25 mg/mL of chitin substrates (α- or β-chitin), 1.0 µM of Sp chitinase (unless stated otherwise) [Sp chiA, Sp chiB, Sp chiC and Sp chiD (GenBank accession no. ABV39247.1, ABV40327.1, ABV42574.1 and ABV41826.1)] were amplified using gene specific primers (Table 2) and cloned, expressed, and purified similarly like Sp cbps (data not shown) and 1.0 mM reduced glutathione. Reaction mixtures were incubated in triplicates at 37°C for 7 days at 1000 rpm in a thermomixer. After every 24 h, 100 µL of reaction mixture was transferred and mixed with 100 µL of 0.02 N NaOH was added to stop the reaction and stored at −20°C until products quantification. Products were quantified by standard chitinase assay as described by Neeraja et al. (2010a, 22).
Supporting Information
Ni-NTA agarose purification of Sp CBPs. Recombinant Sp CBP21, Sp CBP28 and Sp CBP50 were purified using Ni-NTA agarose column chromatography. Elution buffer containing 250 mM imidazole was used to elute Sp CBPs from the column and loaded on 12% SDS-PAGE followed by staining with Coomassie brilliant blue G-250. Lane 1: Protein standards size in kDa indicated to the left, lane 2–4: Purified Sp CBP21, Sp CBP28 and Sp CBP50, respectively.
(DOCX)
Binding of Sp CBPs towards soluble polymeric substrates. Affinity non-denaturing gel electrophoresis was performed at 4°C by preparing 8% polyacrylamide gels. Ten micrograms of Sp CBPs and BSA were electrophoresed without (A), or with 0.1% (w/v) substrates glycol chitin (B), laminarin (C), and CM-cellulose (D). Proteins were visualized by Coomassie blue G-250 staining after electrophoresis. Lane 1: BSA, lane 2–4: Sp CBP21, Sp CBP28 and Sp CBP50.
(DOCX)
Dose-response effects for Sp CBP21 and Sp CBP50 in degradation of β-chitin. Reaction mixture containing 0.25 mg/mL of chitin substrates (α-/β-chitin), 1.0 µM Sp ChiD incubated with different concentrations of Sp CBP21/Sp CBP50 (0.05–0.40 µM) as indicated in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments. (A and B) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP21 and reduced glutathione (RG), (C and D) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP50 and reduced glutathione (RG). Sp CBP21+RG or Sp CBP50+RG: Sp CBP21/Sp CBP50 with reduced glutathione, Sp CBP21+ or Sp CBP50+: only Sp CBP21/Sp CBP50 without reduced gluthathione, Sp CBP21- or Sp CBP50 -: without Sp CBP21/ Sp CBP50 and reduced glutathione.
(DOCX)
Acknowledgments
The authors thank DST-FIST, UGC-SAP and DBT-CREBB for the support to the Department of Plant Sciences, University of Hyderabad. PP thanks the University of Hyderabad for research fellowship, Dr. Daniel van der Lelie, Brookhaven National Laboratory, USA for providing the S. proteamaculans 568, and Dr. Dominique Gillete, Mahatani Chitosan, Veraval, Gujarat, for chitinous substrates.
Footnotes
Competing Interests: The authors have declared that no competing interests exist.
Funding: The work was supported by Department of Biotechnology, Government of India in the form of a research project to ARP; http://dbtindia.nic.in. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Ni-NTA agarose purification of Sp CBPs. Recombinant Sp CBP21, Sp CBP28 and Sp CBP50 were purified using Ni-NTA agarose column chromatography. Elution buffer containing 250 mM imidazole was used to elute Sp CBPs from the column and loaded on 12% SDS-PAGE followed by staining with Coomassie brilliant blue G-250. Lane 1: Protein standards size in kDa indicated to the left, lane 2–4: Purified Sp CBP21, Sp CBP28 and Sp CBP50, respectively.
(DOCX)
Binding of Sp CBPs towards soluble polymeric substrates. Affinity non-denaturing gel electrophoresis was performed at 4°C by preparing 8% polyacrylamide gels. Ten micrograms of Sp CBPs and BSA were electrophoresed without (A), or with 0.1% (w/v) substrates glycol chitin (B), laminarin (C), and CM-cellulose (D). Proteins were visualized by Coomassie blue G-250 staining after electrophoresis. Lane 1: BSA, lane 2–4: Sp CBP21, Sp CBP28 and Sp CBP50.
(DOCX)
Dose-response effects for Sp CBP21 and Sp CBP50 in degradation of β-chitin. Reaction mixture containing 0.25 mg/mL of chitin substrates (α-/β-chitin), 1.0 µM Sp ChiD incubated with different concentrations of Sp CBP21/Sp CBP50 (0.05–0.40 µM) as indicated in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments. (A and B) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP21 and reduced glutathione (RG), (C and D) degradation of α- and β-chitin by Sp ChiD in the presence/absence of Sp CBP50 and reduced glutathione (RG). Sp CBP21+RG or Sp CBP50+RG: Sp CBP21/Sp CBP50 with reduced glutathione, Sp CBP21+ or Sp CBP50+: only Sp CBP21/Sp CBP50 without reduced gluthathione, Sp CBP21- or Sp CBP50 -: without Sp CBP21/ Sp CBP50 and reduced glutathione.
(DOCX)