Morphological and structural characterization of chitin as a substrate for the screening, production and molecular characterization of chitinase by Bacillus velezensis
- Akhil Pilli,
- Digvijay Dahiya,
- Pratap Raja Reddy Chirra,
- Vinay Sreeramula,
- Nitish Venkateswarlu Mogili,
- Seenivasan Ayothiraman
Department of Biotechnology
National Institute of Technology Andhra Pradesh, Tadepalligudem 534101
Environmental Science and Pollution Research 29(57), 86550-86561 · DOI 10.1007/s11356-022-22166-x
Received 15 February 2022 · Accepted 19 July 2022 · Published 27 July 2022
What the study reports
The processing of shellfishery industrial wastes is gaining much interest in recent times due to the presence of valuable components. Chitin is one of the valuable components and is insoluble in most common solvents including water. In this study, a novel gram-positive bacterial strain capable of solubilizing chitin was screened from a prawn shell dumping yard. The chitinolytic activity of the isolated strain was observed through the zone of hydrolysis plate assay. The hyper-producing isolate was identified as Bacillus velezensis through the 16S rRNA sequencing technique. The structural and morphological characterization of raw and colloidal chitin preparation was carried out using FTIR, XRD, and SEM analysis. The residual protein and mineral content, degree of polymerization, and degree of acetylation were reported for both raw and colloidal chitin preparations. There was a linear increase in the chitinase activity with an increase in the colloidal chitin concentration. The maximum activity of chitinase was observed as 38.98 U/mL for the initial colloidal chitin concentration of 1.5%. Supplement of additional carbon sources, viz., glucose and maltose, did not improve the production of chitinase and resulted in a diauxic growth pattern. The maximum chitinase activity was observed to be 33.10 and 30.28 U/mL in the colloidal chitin containing medium with and without glucose as a secondary carbon source, respectively. Interestingly, the addition of complex nitrogen sources has increased the production of chitinase. A 1.95- and 2.14-fold increase in the enzyme activity was observed with peptone and yeast extract, respectively. The chitinase was confirmed using SDS-PAGE, native PAGE, and zymograms. The optimum pH and temperature for chitinase enzyme activity were found to be 7.0 and 44 degrees C, respectively.
Responsible editor Diane Purchase. Corresponding author Seenivasan Ayothiraman, ayothiraman@nitandhra.ac.in.
pH and temperature response of the crude chitinase
Digitised from Fig. 8. The crude extract peaks at 49.73 U/mL at pH 7.0 and 31.95 U/mL at 44 C, with useful activity retained well above mesophilic range.
Activity vs pH
Activity vs temperature, C
Activity response model
Move substrate loading, temperature and pH. The peak reproduces the reported 38.98 U/mL at 1.5% colloidal chitin, pH 7.0 and 44 C; away from the optimum the surface is illustrative.
Reported linear increase in activity with substrate concentration (29.29, 30.53, 38.98 U/mL at 0.5, 1.0, 1.5%).
Optimum 44 C (cited).
Optimum pH 7.0 (cited).
Anchored at the published optimum of the Bacillus velezensis isolate IS2.
Screening of the three chitinolytic isolates
IS1, IS2 and IS3 were recovered from prawn shell dumping yard soil enriched on 1% colloidal chitin. IS2 was the hyper-producer and was carried forward.
Carbon and nitrogen source effects
Glucose and maltose lifted growth rate but not enzyme titre, a catabolite repression signature with diauxic growth. Complex nitrogen sources did the opposite.
| Media composition | Growth rate, 1/h | Activity at 120 h, U/mL | Relative |
|---|---|---|---|
| MM + 0.5% colloidal chitin | 0.174 | 29.29 | |
| MM + 1% colloidal chitin | 0.197 | 30.28 | |
| MM + 1.5% colloidal chitin | 0.219 | 35.76 | |
| MM + 1% chitin + 0.5% glucose | 0.721 | 33.10 | |
| MM + 1% chitin + 0.5% maltose | 0.648 | 27.91 | |
| MM + 1% chitin + 2% peptone | 0.564 | 64.69 | |
| MM + 1% chitin + 2% yeast extract | 0.461 | 58.94 |
Raw versus colloidal chitin
Acid swelling strips protein and mineral, cuts the chain from DP 3715 to DP 592 and raises the measured crystallinity index by removing bound impurities.
| Property | Raw chitin | Colloidal chitin |
|---|---|---|
| Residual protein, % w/w | 23.75 | 6.80 |
| Residual mineral (ash), % w/w | 1.30 | 0.14 |
| Degree of acetylation, FTIR | 39.0% | 45.1% |
| Crystallinity index, XRD | 0.183 | 0.288 |
| Amorphous fraction | 0.817 | 0.712 |
| Average molecular weight | 825 kDa | 132 kDa |
| Degree of polymerization | 3715 | 592 |
| Particle size | < 600 um | < 75 um |
| Swelling index | insignificant | 1 mL |
| RMS roughness Rq | 195.366 | 198.654 |
| Average roughness Ra | 178.703 | 181.753 |
| Skewness Rsk / kurtosis Rku | 1.177 / 1.446 | 1.172 / 1.430 |
FTIR band assignments
Chitin characteristic reflection at 19.5 degrees 2-theta in both samples. Residual protein in raw chitin (23.75%) masked the amine and amide bands, which resolve cleanly after colloidalisation.
Molecular characterization of the enzyme
Native PAGE, zymogram with 0.05% colloidal chitin in the separating gel and SDS-PAGE place a single chitinase near 34 kDa, inside the 20 to 60 kDa band typical of bacterial chitinases.
Native PAGE
Crude enzyme resolved against a protein marker, single dominant band.
Zymogram
Calcofluor White M2R stain after 2 h at 37 C in 0.1 M sodium acetate pH 5.0, one clearance zone confirming a single active chitinase.
SDS-PAGE
4% stacking, 10% separating gel, Coomassie R-250, band at approximately 34 kDa.
How the work was done
- 01
Colloidal chitin preparation
Chitin flakes ground and sieved below 425 um. 150 mL of 12 M HCl added to 10 g flakes and stirred 1 h, undissolved solids removed through muslin, the suspension precipitated into 1 L ice-cold water and held at 4 degrees C for 12 h. Filtered and washed to neutral pH, pressed, foil-wrapped, sterilized and stored at 4 degrees C.
- 02
Substrate characterization
Residual protein by Kjeldahl total nitrogen, mineral content by ashing at 700 degrees C for 1.5 h. FTIR (Jasco), XRD (Bruker D8 Discover, Cu anode, 40 kV, 40 mA) and SEM (Tescan Vega 3 LMU) at 5000x with FIJI ImageJ roughness plots.
- 03
Molecular weight
2 g chitin dissolved in NaOH:urea:water 8:4:88 (% w/w), held at -20 degrees C for 36 h with stirring at 1500 rpm, centrifuged at 4500 rpm for 20 min and filtered at 0.45 um. Intrinsic viscosity by Ubbelohde capillary viscometer, Huggins and Kraemer extrapolation, Mark-Houwink-Sakurada with [eta] = 0.26 M^0.56.
- 04
Enrichment and screening
Soil taken 10-15 cm deep from a prawn shell dumping yard near Bhimavaram, Andhra Pradesh. 3 g added to mineral medium with 1% colloidal chitin (g/L: NH4Cl 3, Na2HPO4 5, KH2PO4 3, CaCl2 2, NaCl 1), 150 rpm, 37 degrees C, 36 h. Three colonies IS1, IS2, IS3 picked and compared; IS2 gave the highest activity and a clear hydrolysis zone after 10 days.
- 05
Identification
Gram staining, motility and carbohydrate tests, then 16S rRNA sequencing (Medauxin, Bangalore) with primers 27F and 1492R, BDT v3.1 chemistry on an ABI 3730xl. ClustalW alignment and a neighbour-joining tree in MEGAX identified the isolate as Bacillus velezensis.
- 06
Chitinase assay
5 mL broth samples centrifuged at 9000 rpm for 10 min; 1 mL crude supernatant incubated with 1 g colloidal chitin for 1 h at 27 degrees C, centrifuged at 5000 rpm for 10 min, reducing sugars by the DNS method (Miller 1959) with blank correction for NAG in enzyme and substrate. Activity = (dC/dt) x Vt / VE x d.
- 07
SDS-PAGE, native PAGE, zymogram
4% stacking and 10% separating gel, Coomassie Brilliant Blue R-250. Zymogram in native PAGE with 0.05% colloidal chitin in the separating gel, incubated in 0.1 M sodium acetate pH 5.0 at 37 degrees C for 2 h, stained with 0.01% Calcofluor White M2R in 0.5 M Tris-HCl pH 8.9 and read under UV.
- 08
Carbon, nitrogen, pH and temperature studies
MM with 0.5, 1 and 1.5% colloidal chitin; MM + 1% chitin with 0.5% w/v glucose or maltose, or 2% w/v yeast extract or peptone. 100 mL cultures at 37 degrees C, sampled for OD600, reducing sugar, Lowry protein and activity. Crude enzyme assayed from pH 3 to 11 and 30 to 50 degrees C.
What it means for the depolymerisation route
An indigenous chitinolytic soil bacterium (IS2) was isolated from a shell waste dumping yard and confirmed by a clear zone of hydrolysis and rising reducing sugar in the broth. 16S rRNA analysis identified it as Bacillus velezensis. Raising colloidal chitin raised chitinase production; glucose or maltose produced diauxic growth without a significant gain, while yeast extract and peptone raised activity 1.95 to 2.14 fold. Native PAGE, zymogram and SDS-PAGE placed the enzyme near 34 kDa, with optima at pH 7.0 and 44 degrees C. Activity at elevated temperature is notable, and purification plus characterization, chitin solubilization and mass transfer in the heterogeneous reaction mixture are the stated next steps toward size-specific chitin oligomers.