Identification and Biochemical Characterization of a Surfactant-Tolerant Chondroitinase VhChlABC from Vibrio hyugaensis LWW-1

Chondroitinases, catalyzing the degradation of chondroitin sulfate (CS) into oligosaccharides, not only play a crucial role in understanding the structure and function of CS, but also have been reported as a potential candidate drug for the treatment of high CS-related diseases. Here, a marine bacterium Vibrio hyugaensis LWW-1 was isolated, and its genome was sequenced and annotated. A chondroitinase, VhChlABC, was found to belong to the second subfamily of polysaccharide lyase (PL) family 8. VhChlABC was recombinant expressed and characterized. It could specifically degrade CS-A, CS-B, and CS-C, and reached the maximum activity at pH 7.0 and 40 °C in the presence of 0.25 M NaCl. VhChlABC showed high stability within 8 h under 37 °C and within 2 h under 40 °C. VhChlABC was stable in a wide range of pH (5.0~10.6) at 4 °C. Unlike most chondroitinases, VhChlABC showed high surfactant tolerance, which might provide a good tool for removing extracellular CS proteoglycans (CSPGs) of lung cancer under the stress of pulmonary surfactant. VhChlABC completely degraded CS to disaccharide by the exolytic mode. This research expanded the research and application system of chondroitinases.


Introduction
Chondroitin sulfate (CS), a kind of glycosaminoglycan that can covalently link with protein to form proteoglycan, is widely distributed in the extracellular matrix and cell surface of animal tissues [1,2]. CS plays an important regulatory role in many physiological processes such as cell development, cell adhesion, proliferation and differentiations [3][4][5][6][7]. Commercially important applications of CS in biological tissue engineering have been explored, which involve combining it with other biopolymers to form scaffolds capable of facilitating and accelerating the regeneration of damaged structures [3].
The CS chain is made up of disaccharide units consisting of D-glucuronic acid (GlcUA)/L-iduronic acid (IdoUA) linked to N-acetyl-D-galactosamine (GalNAc) and sulfated at the C2 position of uronic acid and/or at the C4/C6 position of GalNAc residue [8]. Although the structure of the main chain of polysaccharides is not complex, it shows a high degree of heterogeneity in terms of the degree of sulfation, the sulfation position, and the distribution of the two kinds of different isoguronic acids within the chain. Existing studies have indicated the interaction effect between CS and various growth factors/adhesion alignment of VhChlABC and the identified PL8 family enzymes showed that VhChlABC contained the conserved catalytic residues of the PL8 family in His 484 , Tyr 491 , and Arg 544 ( Figure A1 in Appendix B). The numbers (0~100) on the branches indicate the reliability of the corresponding branches. A larger value means more reliable. Sub, subfamily.

Recombinant Expression and Purification of VhChlABC
The gene vhchlABC was cloned into the vector pET-28a (+) with removal of the signal peptide and addition of (His)6-Tag at both ends. VhChlABC was expressed in soluble forms in the pET-28a (+)/E.coli BL21 (DE3) system. The recombinant VhChlABC was obtained from 40 mL of the bacteria lysate supernatant through nickel affinity chromatography. As shown in Figure 2, a main band (estimated purity > 90%) in SDS-PAGE with a molecular weight of about 110 kDa was identified. The specific activity of purified VhChlABC towards CS-A was 17.54 U/mg. The protein recovery was 16.69% during purification, and about 9.25 mg of purified VhChlABC could be obtained from 1 L of bacterial fermentation broth (Table 1). The numbers (0~100) on the branches indicate the reliability of the corresponding branches. A larger value means more reliable. Sub, subfamily.

Recombinant Expression and Purification of VhChlABC
The gene vhchlABC was cloned into the vector pET-28a (+) with removal of the signal peptide and addition of (His) 6 -Tag at both ends. VhChlABC was expressed in soluble forms in the pET-28a (+)/E.coli BL21 (DE3) system. The recombinant VhChlABC was obtained from 40 mL of the bacteria lysate supernatant through nickel affinity chromatography. As shown in Figure 2, a main band (estimated purity > 90%) in SDS-PAGE with a molecular weight of about 110 kDa was identified. The specific activity of purified VhChlABC towards CS-A was 17.54 U/mg. The protein recovery was 16.69% during purification, and about 9.25 mg of purified VhChlABC could be obtained from 1 L of bacterial fermentation broth (Table 1).  Figure A1 in Appendix B). The numbers (0~100) on the branches indicate the reliability of the corresponding branches. A larger value means more reliable. Sub, subfamily.

Recombinant Expression and Purification of VhChlABC
The gene vhchlABC was cloned into the vector pET-28a (+) with removal of the signal peptide and addition of (His)6-Tag at both ends. VhChlABC was expressed in soluble forms in the pET-28a (+)/E.coli BL21 (DE3) system. The recombinant VhChlABC was obtained from 40 mL of the bacteria lysate supernatant through nickel affinity chromatography. As shown in Figure 2, a main band (estimated purity > 90%) in SDS-PAGE with a molecular weight of about 110 kDa was identified. The specific activity of purified VhChlABC towards CS-A was 17.54 U/mg. The protein recovery was 16.69% during purification, and about 9.25 mg of purified VhChlABC could be obtained from 1 L of bacterial fermentation broth (Table 1).  The A 232 method was used for this detection. Enzyme activity was measured using 0.2% CS-A as substrate (20 mM PB, pH 7.0) under optimal conditions. The volume of the initial fermentation broth was 400 mL, and the volume of the concentrated crude extract was 40 mL.

Substrate Specificity of VhChlABC
The substrate specificity of VhChlABC was determined using a variety of glycolaminoglycans as substrates. As shown in Figure 3, VhChlABC could only degrade CS-A, CS-B, and CS-C, but showed no activity towards other tested substrates. VhChlABC showed the highest degradation activity towards CS-A, and the lowest towards CS-C, which was more than 60% of the former. The specific activity towards CS-A and CS-B was similar. Among the three kinds of CS, VhChlABC possessed the highest k cat /K m value on CS-A at 1.83 min −1 ·mM −1 , followed by CS-C and CS-B (Table 2).
which was more than 60% of the former. The specific activity towards CS-A and CS-B w similar. Among the three kinds of CS, VhChlABC possessed the highest kcat/Km value CS-A at 1.83 min −1 ·mM −1 , followed by CS-C and CS-B (Table 2).  [19,22]. Unlike these, VhChlABC could degrade CS but not H which was also found in the study of ChABC I and ChABC II from Edwardsiella ta LMG2793 and of chondroitin ABC from Bacteroides stercoris [28,29].    [19,22]. Unlike these, VhChlABC could degrade CS but not HA, which was also found in the study of ChABC I and ChABC II from Edwardsiella tarda LMG2793 and of chondroitin ABC from Bacteroides stercoris [28,29].

Effects of Temperature and pH on VhChlABC
According to the results of an optimal temperature test, VhChlABC exhibited the highest activity towards CS-A at 40 • C (Figure 4a), which was similar to most members of PL-8 family, such as ChSase ABC from Bacteroides stercoris and Sphingomonas paucimobilis or ChSase AC from Flavobacterium heparinum et al. [25,29,30]. Thermostability testing indicated that after pre-incubation at a temperature below 30 • C for 1 h, VhChlABC remained about 90% of its initial activity ( Figure 4b). After pre-incubation at 40 • C for 1 h (Figure 4b), the activity retained was more than 60%, and it was approximately 50% after pre-incubation for 2 h (Figure 4c). However, the activity decreased rapidly when the pre-incubation temperature was higher than 40 • C ( Figure 4b). Furthermore, after incubation for more than 2 h at 40 • C, the activity of VhChlABC decreased rapidly and was almost lost until 6 h ( Figure 4c). After treatment at 37 • C for 8 h, VhChlABC could retain about 50% of its initial activity; however, the activity reduced rapidly and was almost lost after 12 h (Figure 4c). In fact, poor thermostability has become an important limiting factor for the application of CS lyases. As reported, cABC I and cABC II from Proteus vulgaris NCTC 4636 cannot maintain 50% of their activity after treatment at 40 • C for 30 min [31]. As for HCDase from Vibrio sp. FC509, its activity gradually declined to 20% after treatment at 30 • C for 4 h [26]. In this research, VhChlABC presented higher stability within 8 h under 37 • C (physiological temperature) and within 2 h under 40 • C (optimal temperature), which might contribute to CS-related clinical application and industrial production.
PL-8 family, such as ChSase ABC from Bacteroides stercoris and Sphingomonas pau or ChSase AC from Flavobacterium heparinum et al. [25,29,30]. Thermostability indicated that after pre-incubation at a temperature below 30 °C for 1 h, Vh remained about 90% of its initial activity (Figure 4b). After pre-incubation at 40 ° (Figure 4b), the activity retained was more than 60%, and it was approximately 5 pre-incubation for 2 h (Figure 4c). However, the activity decreased rapidly when incubation temperature was higher than 40 °C (Figure 4b). Furthermore, after in for more than 2 h at 40 °C, the activity of VhChlABC decreased rapidly and wa lost until 6 h ( Figure 4c). After treatment at 37 °C for 8 h, VhChlABC could reta 50% of its initial activity; however, the activity reduced rapidly and was almost 12 h (Figure 4c). In fact, poor thermostability has become an important limiting f the application of CS lyases. As reported, cABC I and cABC II from Proteus vulgar 4636 cannot maintain 50% of their activity after treatment at 40 °C for 30 min [31 HCDase from Vibrio sp. FC509, its activity gradually declined to 20% after treatm °C for 4 h [26]. In this research, VhChlABC presented higher stability within 8 h °C (physiological temperature) and within 2 h under 40 °C (optimal temperature might contribute to CS-related clinical application and industrial production. The optimal pH value for the degradation activity of VhChlABC against C pH 7.0 in 50 mM Na2HPO4-NaH2PO4 buffer (Figure 5a). Moreover, VhChlABC co stable in different buffers of pH 5.0~10.6 ( Figure 5b). With the high stability VhChlABC might be easier to preserve and more widely used in the industry. The optimal pH value for the degradation activity of VhChlABC against CS-A was pH 7.0 in 50 mM Na 2 HPO 4 -NaH 2 PO 4 buffer (Figure 5a). Moreover, VhChlABC could keep stable in different buffers of pH 5.0~10.6 ( Figure 5b). With the high stability on pH, VhChlABC might be easier to preserve and more widely used in the industry.

Effects of Metal Ions, Chelators, and Surfactants on VhChlABC
As shown in Figure 6a, no tested metal ions enhanced the CS degradation activity of VhChlABC significantly. Zn 2+ , Co 2+ , and Ni 2+ could strongly inhibit its activity, which was consistent with Vibrio sp. FC509 HCDase and HCLase-related results [26,27]. The structural changes caused by the affinity of heavy metals towards the SH, CO, and NH groups in the amino acids might be the crucial factor for the inhibition of VhChlABC by metal ions such as Zn 2+ [32]. In addition, differences of amino acids on the protein surface might also be important for the metal ion effect [33], which might result in the different effects of metal ions on HCDase, HCLase, and VhChlABC. Additionally, NaCl enhanced the CS-degradation activity of VhChlABC at low concentrations (≤0.25 M) and exhibited the maximum effect at 0.25 M (Figure 6b). Notably, even if there was no NaCl in the reaction system, VhChlABC still maintained nearly 50% of its maximum activity ( Figure  6b), which indicated that VhChlABC had no dependency on NaCl and could satisfy more NaCl-limited production conditions.
In particular, VhChlABC well tolerated both SDS and Tween-20, maintaining more than 50% of its maximum activity in the presence of up to 10% (w/v) SDS and having almost no loss of activity in the presence of up to 10% (v/v) Tween-20 ( Figure 6c). Although research has continued to report glycosaminoglycanases (GAGases) of novel structures and functions, studies on surfactant-tolerant GAGases are almost nonexistent. Besides being used as detergents in daily life, other applications of surfactants could cover almost all fine chemical fields [34]. Unfortunately, most of the reported GAGases were inactivated in the presence of very low concentrations of SDS. As reported, SDS could almost completely inhibit the activity of enCSase from Photobacterium sp. QA16 [20], Vpa_0049 from Vibrio sp. QY108 [35], and ChSase ABC from Acinetobacter sp. C26 [24], with final concentrations of 5 mM, 1 mM, and 5 mM, respectively. Unfortunately, the surfactant tolerance of many enzymes has not been measured, including HCDase from Vibrio sp. FC509. The crucial domains and key amino acid residues that determine the surfactant tolerance of VhChlABC must be further explored.
It was considered a safe and effective strategy to remove extracellular CS proteoglycans (CSPGs) using chondroitinase, which was reported to promote viral spread and infection in oncolytic virus (OV)-mediated treatment of astrocytomas [36]. However, the sensitivity of chondroitinase to trace surfactants might limit its application in the lung

Effects of Metal Ions, Chelators, and Surfactants on VhChlABC
As shown in Figure 6a, no tested metal ions enhanced the CS degradation activity of VhChlABC significantly. Zn 2+ , Co 2+ , and Ni 2+ could strongly inhibit its activity, which was consistent with Vibrio sp. FC509 HCDase and HCLase-related results [26,27]. The structural changes caused by the affinity of heavy metals towards the SH, CO, and NH groups in the amino acids might be the crucial factor for the inhibition of VhChlABC by metal ions such as Zn 2+ [32]. In addition, differences of amino acids on the protein surface might also be important for the metal ion effect [33], which might result in the different effects of metal ions on HCDase, HCLase, and VhChlABC. Additionally, NaCl enhanced the CS-degradation activity of VhChlABC at low concentrations (≤0.25 M) and exhibited the maximum effect at 0.25 M (Figure 6b). Notably, even if there was no NaCl in the reaction system, VhChlABC still maintained nearly 50% of its maximum activity (Figure 6b), which indicated that VhChlABC had no dependency on NaCl and could satisfy more NaCl-limited production conditions.
In particular, VhChlABC well tolerated both SDS and Tween-20, maintaining more than 50% of its maximum activity in the presence of up to 10% (w/v) SDS and having almost no loss of activity in the presence of up to 10% (v/v) Tween-20 (Figure 6c). Although research has continued to report glycosaminoglycanases (GAGases) of novel structures and functions, studies on surfactant-tolerant GAGases are almost nonexistent. Besides being used as detergents in daily life, other applications of surfactants could cover almost all fine chemical fields [34]. Unfortunately, most of the reported GAGases were inactivated in the presence of very low concentrations of SDS. As reported, SDS could almost completely inhibit the activity of enCSase from Photobacterium sp. QA16 [20], Vpa_0049 from Vibrio sp. QY108 [35], and ChSase ABC from Acinetobacter sp. C26 [24], with final concentrations of 5 mM, 1 mM, and 5 mM, respectively. Unfortunately, the surfactant tolerance of many enzymes has not been measured, including HCDase from Vibrio sp. FC509. The crucial domains and key amino acid residues that determine the surfactant tolerance of VhChlABC must be further explored. industry [37]. The identification of the surfactant-tolerance gene vhchlABC is of great significance for understanding the stress tolerance mechanism of Vibrio.

Degradation Mode and End Products of VhChlABC
SEC was performed to analyze the degradation modes and end products of CS-A, CS-B, and CS-C by VhChlABC. Per the results, only one major product peak was detected during the reaction (Figure 7a), indicating that VhChlABC degraded CS through the exolytic mode, which was consistent with HCDase [26]. All the enzymatic properties suggested that VhChlABC was an EC 4.4.2.21-chondroitin-sulfate-ABC exolyase. The end products of CS-A, CS-B, and CS-C were all identified as the same single peak (data It was considered a safe and effective strategy to remove extracellular CS proteoglycans (CSPGs) using chondroitinase, which was reported to promote viral spread and infection in oncolytic virus (OV)-mediated treatment of astrocytomas [36]. However, the sensitivity of chondroitinase to trace surfactants might limit its application in the lung environment under the stress of pulmonary surfactant. Therefore, the high surfactant tolerance of VhChlABC might open a new horizon for the treatment of lung cancer. In addition, Vibrio is widely distributed in different water bodies and marine animals, has strong vitality, and has been recognized as an important pathogen in the aquaculture industry [37]. The identification of the surfactant-tolerance gene vhchlABC is of great significance for understanding the stress tolerance mechanism of Vibrio.

Degradation Mode and End Products of VhChlABC
SEC was performed to analyze the degradation modes and end products of CS-A, CS-B, and CS-C by VhChlABC. Per the results, only one major product peak was detected during the reaction (Figure 7a), indicating that VhChlABC degraded CS through the exolytic mode, which was consistent with HCDase [26]. All the enzymatic properties suggested that VhChlABC was an EC 4.4.2.21-chondroitin-sulfate-ABC exolyase. The end products of CS-A, CS-B, and CS-C were all identified as the same single peak (data not shown), with a molecular weight of 458.06 m/z (z = 1) identified by negative-ion ESI-MS (Figure 7b)

Isolation of Marine CS Lyase-Producing Bacteria
Seawater sample was collected from Zhanqiao, QingDao, China. A selective medium, which consisted of 0.3% (w/v) KH 2 PO 4 , 0.7% (w/v) K 2 HPO 4 ·3H 2 O, 0.2% (w/v) (NH 4 ) 2 SO 4 , 0.01% (w/v) FeSO 4 ·7H 2 O, 3% NaCl, 0.05% (w/v) CS (CS-A: CS-B: CS-C = 1:1:1 by mass), and 1.5% (w/v) agar (pH 7.0), was used to isolate CS lyase-producing bacteria from the seawater sample. Obviously, CS was the sole carbon source. The screening model referred to the Gram's iodine plate assay method for hyaluronidase [38]. Briefly, after pre-incubation at 25 • C for 48 h, the plates containing the microorganisms were soaked with Gram's iodine for 1 min. Clones with distinct clearance zones were detected as CS-degrading strains and then purified with fresh selective medium plates three times to obtain the pure cultured strains. Then, the pure monoclonal strains were cultured in 100 mL marine broth 2216 at 25 • C and 160 rpm/min for 48 h, and the CS lyase activity in the culture supernatant was determined using the A 232 method (as shown in Section 3.6). The strain LWW-1, which exhibited the highest CS lyase activity, was obtained and used in the following experiment.

Sequence Analysis of VhChlABC
The genomic DNA of strain LWW-1 was prepared using Tianamp bacteria DNA kit. The genome of strain LWW-1 was sequenced by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) and then annotated with Rapid Annotation using Subsystem Technology (RAST). Sequence similarity analysis was performed by online BLAST. The phylogenetic tree was drawn using the neighbor-joining method by MEGA 7.1. The molecular weight (Mw) and isoelectric point (pI) were analyzed by the compute Mw/pI tool on ExPASy of Swiss Bioinformatics Resource Portal (http://us.expasy.org/tools/pi_ tool.html accessed on 15 November 2020). The SignalP 5.0 server (http://www.cbs.dtu.dk/ services/SignalP/ accessed on 15 November 2020) was used to predict the signal peptide and its cleavage site in VhChlABC. The protein domains within VhChlABC were identified using the Conserved Domains server of NCBI database (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi accessed on 15 November 2020). Amino acid alignment with other enzymes of the PL8 family was carried out using ESPrit 3.0 (https://espript.ibcp.fr/ ESPript/ESPript/ accessed on 15 November 2020).

Recombinant Expression and Purification of VhChlABC
The full-length gene of vhchlABC without the signal peptide sequence was amplified with super-fidelity DNA polymerase (Vazyme, Nanjing, China) and the primer pairs ChlABC-F (GTACcatatgAGCGAAAATGTTGAAAGCA) and ChlABC-R (GTACctcgag-TACTTTTTTCAGCATGAATTTT), taking the genomic DNA of V. hyugaensis LWW-1 as the template. After digestion with Nde I and Xho I, the gel-recovered PCR products were ligated into plasmid pET-28a (+). Then, the plasmid pET-28a-VhChlABC was transformed into E. coli BL21 (DE3) cells to obtain the recombinant expression strains.
The recombinant expression strains were cultured in Luria-Bertani (LB) medium (containing 30 µg/mL kanamycin) with shaking at 37 • C until the OD 600 value reached 0.4~0.6. Then, it was induced to express VhChlABC by adding 0.1 mM isopropyl-1thio-β-D-galactoside (IPTG) and incubated at 18 • C for 24 h. After centrifugation with 8000 rpm/min for 30 min at 4 • C, cells were resuspended in precooled 20 mM phosphate buffer (PB) containing 500 mM NaCl at pH 7.0 (binding buffer) at a volume of 1/10 of the initial fermentation broth, then ruptured by the high-pressure cell cracker at 4 • C. Next, the supernatant (usually named crude extract) containing the soluble target protein was collected by centrifugation at 12,000 rpm/min for 30 min. The crude extract was subsequently filled by a constant current pump onto a Ni-Sepharose column (GE Healthcare, Pittsburgh, PA, USA) pre-balanced with binding buffer. The (His) 6 -tagged VhChlABC protein was eluted from the column using binding buffer with 300 mM imidazole added. The molecular weight and purity of VhChlABC were examined by SDS-PAGE combined with Coomassie Bright Blue staining. Protein content was measured according to the instructions of BCA protein assay kit (EpiZyme, Shanghai, China).

Enzyme Activity Determination of VhChlABC
In this study, two methods were used to determine the enzyme activity. As for the A 232 method [28], a reaction system containing 100 µL of enzyme sample, which in turn contained approximately 1.0 µg of purified VhChlABC, and 900 µL of 0.2% (w/v) CS substrate was incubated at 40 • C for 10 min. The change of absorbance value at 232 nm was measured to quantify the unsaturated double bonds in the system. One unit (U) was defined as the amount of enzyme required to catalyze the production of 1 µmol of 4,5-unsaturated uronic acid per minute. The millimolar absorption coefficients for CS-A, CS-B, and CS-C were 5.1, 5.1, and 5.5 respectively [39].
As for the DNS (dinitrosalicylic acid) method [40], 50 µL of enzyme sample, which contained approximately 3.0 µg of purified VhChlABC, and 450 µL of 0.2% (w/v) CS substrate (20 mM PB, pH 7.0) were co-incubated at 40 • C for 10 min. After that, 375 µL of DNS reagent, prepared as described in Appendix A, was added to the system and boiled at 100 • C for 10 min. Then, the reducing sugar content was quantified by measuring the absorbance value of the reaction solution at 520 nm using D-glucosamine as a standard. For both methods, the same reaction with an equal amount of enzyme, inactivated by boiling for 10 min, was used as the blank. One unit was defined as the amount of enzyme that generated reducing sugars corresponding to 1 µmol of glucosaminehydrochloride per minute under standard conditions.

Substrates Specificity Analysis of VhChlABC
Various commercial polysaccharide substrates at 0.2% (w/v), including HA, CS-A, CS-B, CS-C, alginate, pectin, xanthan and heparin (20 mM PB, pH 7.0), were used as substrates to determine the substrate specificity of VhChlABC. A 232 method was used for this test under optimal conditions.

Biochemical Characterization of VhChlABC
Biochemical characterization was determined using CS-A as the substrate. The A 232 method was used for this test. For optimum temperature detection, 0.2% (w/v) CS-A (20 mM PB, pH 7.0) was digested by VhChlABC at a serial temperature gradient from 0 to 60 • C. To determine the thermostability, VhChlABC was pre-incubated at different temperatures (0~50 • C) for 1 h, and the residual activity was measured at 40 • C. Furthermore, the residual activity of VhChlABC was examined at different times post incubation at 37 • C and 40 • C. The initial specific activity detected at 40 • C was set as 100%.
For optimum pH detection, the activity of VhChlABC was analyzed under different pH buffers of 50 mM at 40 • C, including Na 2 HPO 4 -NaH 2 PO 4 with pH of 6.0~8.0, Tris-HCl with pH of 7.05~8.95, NaH 2 PO4-citric acid with pH of 3.0~8.0, and glycine-NaOH with pH of 8.6~10.6. To analyze the effect of pH on stability, VhChlABC was incubated in a series of pH buffers at 4 • C for 12 h, respectively. Then, residual activity was measured by the A 232 method under optimal conditions. Different ions, including K + , Li + , Zn 2+ , Ca 2+ , Ba 2+ , Mn 2+ , Mg 2+ , Co 2+ , and Ni 2+ , and chelator (EDTA) were separately added to the reaction mixture at a final concentration of 1 mM to detect their effect on CS degradation by VhChlABC. NaCl dependence might be the specificity of marine active enzymes; therefore, the effects of NaCl concentrations (0~1.0 M) on the activity of VhChlABC were detected in this research. The A 232 method was used for the above detection under optimal conditions. The effects of SDS and Tween-20 on VhChlABC were detected by adding different final concentrations of SDS (0~10%, w/v) and Tween-20 (0~10%, v/v) separately into the reaction system. SDS and Tween-20 can greatly increase the absorbance of the substrate at 232 nm, which is beyond the reliable range of the A 232 method. Therefore, the DNS method was used for this detection.

Reaction Kinetics of VhChlABC towards CS
CS-A, CS-B, and CS-C were each dissolved in 20 mM PB (pH 7.0) to prepare substrates with concentrations from 0.2 to 2.0 mg/mL. The absorbance value of the enzyme-substrate (1:9 by vol) reaction system at 232 nm was measured after incubation at 40 • C for 3 min. V max and K m values were calculated using the Michaelis-Menten equation and the curve fitting program by the non-linear regression analysis using Graphpad Prism 8. The k cat value was the ratio of V max to the enzyme concentration.

Reaction Mode and End Products of VhChlABC
The reaction modes of VhChlABC towards CS-A, CS-B, and CS-C were monitored. Degradation reaction was initiated by uniformly mixing enzyme and substrate (20 mM PB, pH 7.0) in proper proportions under optimal conditions and terminated by boiling at different timepoints. The unsaturated oligosaccharide products with different degrees of polymerization in the samples of different timepoints were separated and analyzed by size-exclusion chromatography (SEC). Fast protein liquid chromatography (FPLC, GE Healthcare, Pittsburgh, PA, USA) combined with a Superdex peptide 10/300 GL column (GE Healthcare, Pittsburgh, PA, USA) was used for this process. For the mobile phase, 0.2 M NH 4 HCO 3 at a flow rate of 0.2 mL/min was used. The absorbance value at 232 nm was monitored.

Conclusions
In this study, a novel PL-8 chondroitin sulfate lyase, VhChlABC, was identified and characterized from marine bacterium V. hyugaensis LWW-1. Although VhChlABC has the highest amino acid identify (82.05%) with HCDase, it possesses some significantly different characteristics from the latter. The discovery of surfactant-tolerant chondroitinase provides a new way for the treatment of CS-related diseases in special pathological conditions. The wider pH tolerance range makes VhChlABC a potentially good enzymatic tool in industry application without rigorous reaction and storage conditions.