Bioconversion of chitin and concomitant production of chitinase and N-acetylglucosamine by novel Achromobacter xylosoxidans isolated from shrimp waste disposal area

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket–Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition—arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.


Materials and methods
colloidal chitin preparation. About 5 g of chitin was mixed with 30 ml of HCl acid (35.5%) and incubated overnight at 4 °C. The colloidal chitin was precipitated by slowly adding 250 ml of chilled ethanol (50%), with constant stirring at 4 °C and left for overnight. The colloidal chitin was centrifuged at 10,000 g for 20 min, and sterile distilled water was used to wash the pellets until the pH value is neutral. isolation of chitinase producing bacteria. The soil samples were collected from five different places of the shrimp and crab waste disposable area near Port Novo landing centre situated on the south-east coast of India (Parangipettai-Lat. 11° 29′; long. 79 o 46′ E). The 1 g of soil sample was serially diluted and plated on chitin minimal agar plates (Colloidal chitin, 1%; K 2 HPO 4, 0.3 g; NaCl, 4 g; KH 2 PO 4, 0.3 g; MgSO 4 ·7H 2 O, 0.5 g; Agar, 1.5% per litre). The plates were incubated at 30 °C for 3 days. The bacterial colony showing the maximum zone of chitin clearance was selected for further studies.
Scientific RepoRtS | (2020) 10:11898 | https://doi.org/10.1038/s41598-020-68772-y www.nature.com/scientificreports/ chitinase assay. 2.5 ml of 1% colloidal chitin (substrate) was mixed with 2.5 ml of PBS, and then 0.5 ml crude enzyme was added; next, the flask was incubated at 45 °C for 1 h. The reaction mixture was added with dinitrosalicylic (DNS) acid and was placed within a boiling water bath for 10 min to stop the reaction. The supernatant was collected by centrifuge, and the reduced sugar was estimated at 540 nm (UV spectrophotometer). Using the GlcNAc standard curve, under assay condition, the quantity of enzyme that yields 1 µmol of reducing sugar/ minute can be defined as one unit of chitinase.
characterisation of isolates. The biochemical and morphological characteristics of chitin degrading isolates were characterised, and the strains were identified using Bergey's manual of determinative bacteriology 20 . The potential bacterial strain was also characterised by 16S rDNA sequencing.
Molecular characterisation. Genomic  phylogenetic analysis. The Genei PCR purification kit (Genei, Bangalore, India) was used in the purification of the amplified PCR products. The sequence of 16s rDNA amplified product was obtained by an automated sanger sequencer (Bioserve, Hyderabad, India). The sequence was edited using BioEdit ver. 7.9 22 followed by a BLAST search in the NCBI public database to identify the closest sequences. The Phylogenetic tree was drawn in the Mega 6.0 version using the neighbour-joining method 23 . The partial 16S rDNA sequence of bacterial strain was deposited in the GenBank database, and accession numbers were received upon submission to the NCBI database.
Extraction and purification of chitinase. The crude enzyme was obtained by centrifuging the production medium at 8000 g for 10 min, followed by precipitation of enzymes at different concentration of [50-80% (w/v)] ammonium sulfate slowly added into the supernatant under constant stirring for 30 min at 4 °C. Next, the protein precipitate was collected by centrifugation at 10,000 g for 30 min at 4 °C and dissolved in Tris-HCl buffer with pH.7.5. Next, the enzymes were dialysed at 4-6 °C against 20 mM Tris-HCl buffer with pH 7.5 and incubated for 24 h, and the buffer was changed at every 8 h interval. The dialysed enzyme solution was then purified with Sephadex 75 column (20 cm × 1.5 cm) equilibrated with 20 mM Tris-HCl buffer with the pH 8.0. The flow rate of 1 ml/5 min was used to elute the enzyme. The eluted fractions were estimated for the chitinase activity. The fractions showing chitinase activity were mixed together and concentrated by using a fast flow DEAE-Sepharose column (1.6 cm × 20 cm) pre-equilibrated with 20 mM Tris-HCl of pH 8 (10 mmol/l). The bounded chitinase was eluted at a flow rate of 400 µl/min using different gradients of NaCl buffer (0-0.5 mol/l) at 4 °C. The collected chitinase fractions were used as an enzyme source for chitinase assays and protein quantification 24 . Determination of optimal temperature and pH for enzyme activity. The reaction mixture was incubated at different temperatures and tested for chitinase activity to find the optimum temperature for enzyme activity. To determine the thermal stability of the enzyme, 50 mM Tris-HCl buffer-pH 9.0 along with 0.2 ml of the purified enzyme was incubated for 1 h at a temperature range of 35-95 °C with 5 °C interval. The enzyme activity was evaluated by withdrawing the aliquots for every 15 min and tested using standard chitinase assay. To determine the optimal pH for enzyme activity, 0.2 ml of the purified enzyme along with 50 mM Phosphate buffer was incubated at different pH ranging between 4 and 9 and the residual activity was measured using chitinase assay.
Identifying the significant variables for maximum chitinase production. The medium components for maximising the chitinase production by A. xylosoxidans were screened by using Plackett-Burman design (PBD) 18 . The medium components chosen for the present study include colloidal chitin, yeast extract, peptone, KH 2 PO 4 , FeSO 4 , ZnSO 4 and MgSO 4 , with each component being represented at two levels, low (− 1) to high (+ 1). Table 1 shows the experimental design. The culture medium was prepared accordingly and incubated at 37 °C for 3-5 days. After incubation, the culture filtrate was centrifuged at 8000 g for 15 min. The actual (measured) and estimated (PBD-based) enzyme values are given in Table 1 (Table 1), were further optimised using RSM, i.e., we employed a central composite design (CCD) for maximum production of chitinase enzyme. The significant variables for chitinase production, i.e. peptone, chitin, KH 2 PO 4 and MgSO 4 and a total of 31 experiments were performed with these variables at five different levels (− 2, − 1, www.nature.com/scientificreports/ 0, + 1, and + 2). The full experimental plan, including the values of the significant variables, is given in Table 2.
The enzyme activity U/ml (response value) was obtained by calculating the average of the triplicate in each trial. Analysis of variance (ANOVA) was performed on the data obtained from RSM on Chitinase production. The response surface regression involves the polynomial equation (2): where Y is the prediction, β 0 is the constant term, β i is the ith linear coefficient, β ii is the ith quadratic coefficient, β ij is the ijth interaction coefficient, and X i , X j are independent variables. The independent variables were expressed as X1, X2, X3, X4, X5 and can be represented as the second-order polynomial equation (3): Fisher's check was used to evaluate the statistical importance of the equation version and model terms. The quality of fit for the second-order polynomial equation was expressed through the coefficient of determination (R 2 ) and the adjusted R 2 . The fitted polynomial equation (3) was expressed as 3-dimensional surface plots to visualise the relationship between the responses and the utilised variables. Point optimisation approach was deployed to optimise each variable for the maximum reaction 25 .
Amino acid analysis. The colloidal chitin was mixed with 500 µl of purified chitinase and incubated at 45 °C for 1 h. Subsequent to incubation, the centrifugation of the reaction mixture was performed at 3500 g for 15 min. 1 N NaOH was used to neutralise the filtered supernatant. The neutralised supernatant was diluted to 100 times of the volume by adding milli-Q water, and 20 µl was injected into HPLC (Shimadzu, USA) containing LC1oAT HPLC pumps. The analysis was carried out with 30% milli-Q water and 70% acetonitrile at a flow rate of 1000 µl/min in 40 °C as mobile phase and detected by SPD-M10A detector with UV absorbance at 250 nm. ethical approval. No animal or human studies were carried out by the authors.

Results and discussion
isolation and characterisation of chitin producing strains. In this present investigation, the isolation and characterisation of potent chitin degrading bacteria from the marine environment were performed. Fourteen bacterial strains were isolated from the shrimp waste disposal site, among the 14 strains one bacterial strain showing maximal chitinase activity (1.8 U/ml, Fig. 1) was selected, and this strain was named as chitinase producing bacterial strain 4 (CHI4). The morphological and biochemical test showed this strain CHI4 was rodshaped, Gram-negative, motile bacteria, utilising catalase and oxidase and oxidise sugars such as xylose and glucose. The 1091 bp 16S rDNA sequence and blast analysis showed that this bacterial strain was closely similar to Achromobacter xylosoxidans. Moreover, 99 per cent similar to the Achromobacter xylosoxidans strain Osb (Genbank accession no: MN889379.1) and Achromobacter xylosoxidans strain Spa05NA (Genbank accession no: MT052658.1). The accession number in the NCBI database for the deposited 16S rDNA partial sequence was JQ756451. The phylogenetic tree for the selected strains was constructed using the neighbour-joining method (Fig. 2). Earlier, this species was named as Alcaligenes xylosoxidans. Later, based on the 16S rDNA sequence, it was renamed to Achromobacter xylosoxidans 26 . It is widely found in soil, water, dialysis and chlorhexidine solutions, sometimes present in the gastrointestinal and respiratory tract of humans 27 .
In the past, numerous marine bacterial strains were reported to produce chitinase such as Bacillus cereus SV1 28 , Alteromonas sp. O-7 29 , Aeromonas hydrophila 30 , Pseudoalteromonas sp. DC14 31 , Pseudoalteromonas sp.  Chitinase purification. The chitinase enzyme was effectively precipitated from the CHI4 culture supernatant system by salting-out method at 75% concentration of ammonium sulfate. After the dialysis and Sephadex-75 column chromatography purification, seven fractions containing enzyme activity were obtained. The enzyme specific activity increased to 10.75 U/mg after threefold purification using the Sephadex column, and the protein recovered was 8 mg (Table 3) and a total activity of 86 U/mg (Yield-12%) of chitinase enzyme were obtained. However, a high amount of recovery rate with 27% yield was noted in Vibrio sp. 36 .
Effect of temperature and pH on the stability and activity of chitinase. The purified chitinase enzyme obtained from A. xylosoxidans was active 100% at an optimum temperature of 45 °C. The enzyme activity was observed at temperatures above 45 °C, but it decreased to 45% at 50 °C and lost its activity above 65 °C (Fig. 3). The optimum temperature for chitinase activity in various marine bacteria was reported in the past, i.e. 55 °C for Bacillus cereus SV1 28 , 50 °C for Alcaligenes xylosoxydans 35 7 . However, in the present study, the optimum temperature for chitinase activity ranged between 30-50 °C and was able to withstand up to 65 °C. Chitinase from A. xylosoxidans was active in pH ranging between 7 and 9 and was found to have an optimum pH of 8.0. Moreover, the enzyme was 83% stable and 80% active at pH 8.0 (Fig. 4). The activity of chitinase reduced at pH beyond and below 6 due to the changes of ionic residues in the protein molecules. The ionic amino acid residue, which is found at the active site of the protein, is critical to maintaining protein conformation structure. Our results compared with the chitinase produced by the Alteromonas sp. O-7 29 . Also, the Streptomyces chilikensis RC1830 produced chitinase was active between pH 5-8 with an optimum pH 7 37 . The activity of chitinase produced from certain marine bacterias like Sphingomonas sp. CJ-5 33 and Serratia marcescens 7 has an optimum pH of 7, and the optimum pH was 9 for Pseudoalteromonas sp. DC14 31 . Whereas, chitinase of Alcaligenes xylosoxydans was active in acidic pH 35 and herein, A. xylosoxidans chitinase was active in a wide range of pH from neutral to base with 80% activity.

Screening of significant variables by Plackett-Burman design.
Totally twelve variables were selected for analysing the effects on chitinase production by A. xylosoxidans using the Plackett-Burman design. The variables showing a positive impact on the enzyme production were selected for the optimisation study, and the significant variables of chitinase production with corresponding responses are exposed in Table 1 5 and Table 4). Significant variables were identified, i.e., Colloidal chitin, peptone, KH 2 PO 4 and MgSO 4 were selected along with the incubation period (37 °C for 3-5 days) to determine the optimum chitinase production by CCD further. Similar experimental variables such as yeast extract, urea, KH 2 PO 4 and colloidal chitin, were found to influence chitinase production by Paenibacillus sp. 38 , however, in the present study peptone showed a positive impact. Colloidal chitin, syrup of date, yeast extract, K 2 HPO 4 and KH 2 PO 4 showed a positive impact on chitinase production in Streptomyces griseorubens C9 39 . The colloidal chitin as the sole carbon source, showed enhanced chitinase production in Microbispora sp. 40 . In contrast, better chitinase production was observed in the marine bacteria, Aeromonas hydrophila SBK1 when shrimp shell powder was used rather than colloidal chitin 41 . Phosphate was reported to have a negative effect on the chitinase production in Streptomyces griseus 42 , but herein, it did have a modest positive effect on chitinase production (K 2 HPO 4 ).
Peptone and yeast extract, the nitrogen sources (organic) did increase our chitinase production. Similar to the studies demonstrating chitinase production from Alcaligenes xylosoxydans 34 , Serratia marcescens 43 and Paenibacillus sp. D1 38 . In contrast to this, Paenibacillus sp. AD showed maximum chitinase synthesis when ammonium sulfate was used as a nitrogen source 44 , while sodium nitrate was ideal for the production of chitinase in Stachybotrys elegans 45 . In the present study, KH 2 PO 4 and MgSO 4 showed a positive impact on chitinase production, whereas, their influence was insignificant during chitinase production by Pseudomonas fluorescens strain HN1205 46 .   Table 5, and the P value for the model was 0.0001, P value of lack of fit was 0.260, suggesting that the data was a good fit with the model. The three-dimensional response surface plots (regression equation in graphical representations) are shown in Figs. 6, 7, 8, 9, 10 and 11. The primary objective of the response surface is to ensure the variables' optimum values response is maximised powerfully. The surface plots affirm that the objective function was unimodal,  www.nature.com/scientificreports/ which showed an optimum in the centre, and that the model is in a good prediction of the experiment. The main optimum point was estimated by the gradient method with the direction of the medium was the steepest rise for the chitinase production and estimated from the surface plots. The optimal values of peptone, chitin, KH 2 PO 4 and MgSO 4 were estimated in actual units, and they were 0.5545 g/l, 30.6061 g/l, 0.50 g/l and 0.2818 g/l, respectively, with a chitinase activity of 464.2 U/ml (predicted). To confirm the predicted values, experiments   www.nature.com/scientificreports/ were performed with these predicted conditions, and the recorded chitinase activity was 467 U/ml (Fig. 12). This activity was slightly high when compared to the predicted value. Such studies reiterate the fact that CCD is a highly reliable model. Furthermore, the recorded chitinase activity was also higher than the chitinase production observed in most marine bacteria isolated from shrimp shell waste. A review on chitinase production and the produced quantity   www.nature.com/scientificreports/ by isolated marine bacterial strains can be seen in Table 6. Whereas, one should note that chitinase production from the marine fungi Aspergillus flavus MK20 isolated from coastal sediment can produce chitinase up to 620.5 U/ml, with RSM (Box-Behnken Design-BBD) based estimation up to 1520 U/ml 47 . Optimisation of Paenibacillus sp. D1, a marine microbial produced chitinase was demonstrated using CCD (predicted 85.8 U/ml) with an experimental design including 30 different formulations yielded 93.2 U/ml 38 . Whereas, herein, the maximum similarity was recorded between the experimental and the predicted results. Similar studies have also reported a positive effect of using statistical methods such as CCD to increase chitinase production by 141% in Alcaligenes xylosoxidans 35 . Amino acid analysis of chitin degraded product. The chitin degraded end products were analysed by HPLC. Usually, the chitin derivatives are oligomeric and monomeric polysaccharides, and few proteins are present. The results showed that in total, 15 amino acids were present ( Fig. 13 and Table 7). The primary amino acid profile composition of the chitin degraded products includes lysine, aspartic acid, arginine, and low levels of isoleucine and methionine. Presence of major amino acids within the derivatives makes it an ideal aquaculture supplement.
While most prior studies focused on the amino acid profile of the chitinase enzyme, we have investigated the amino acid profile of chitin degraded products. Which lead to an exciting find that the amino acid profile of chitin degraded products has more composition along with traces of chitinase when compared with the amino acid profile of chitinase enzyme. Likewise, asparagine, threonine, glycine and alanine were reported as the major amino acids in chitin derivatives 48 .  Table 6. Review on chitinase production and the recorded quantity by isolated marine bacterial strains.

Source of isolation Quantity in U/ml RSM-quantity in U/ml
Streptomyces sp. 49  In conclusion, we isolated 14 different strains of marine bacteria capable of degrading chitin. Out of that, the bacterial strain that had high chitinase activity was isolated. We have demonstrated that this Achromobacter xylosoxidans strain isolated from a shrimp waste disposal site can produce chitinase, which in turn can degrade chitin along with the concomitant production of N-acetyl-d-glucosamine. The simultaneous production of value-added bioproducts makes this an eco-friendly solution for environmental pollution caused by shrimp waste disposable. Moreover, these end products could also be promoted as a cost-effective supplement for the aquaculture industry. Furthermore, we have also demonstrated that this chitinase production by Achromobacter xylosoxidans can be optimised using RSM techniques.