Robust chitinolytic activity of crab-eating monkey (Macaca fascicularis) acidic chitinase under a broad pH and temperature range

Diet of the crab-eating monkey (Macaca fascicularis) consists of both plants and animals, including chitin-containing organisms such as crabs and insects. This omnivorous monkey has a high expression of acidic chitinase (CHIA) in the stomach and here, we report on its enzymatic properties under different conditions. When we compared with Mus musculus CHIA (Mm-CHIA), Macaca fascicularis CHIA (Mf-CHIA) exhibits higher chitinolytic activity at broad pH (1.0–7.0) and temperature (30–70 ℃) range. Interestingly, at its optimum pH (5.0), Mf-CHIA showed the highest activity at 65 °C while maintaining it at robust levels between 50 and 70 °C. The degradation efficiency of Mf-CHIA was superior to Mm-CHIA toward both polymeric chitin as well as an artificial chromogenic substrate. Our results show that unique features of Mf-CHIA including its thermostability warrant the nomination of this enzyme for potential agricultural and biomedical applications.

www.nature.com/scientificreports/ concentrations 34 . Here, we used 4-NP-(GlcNAc) 2 and compared its degradation at concentrations ranging from 20 μM to 400 μM. The substrate degradation intensified with its increasing initial concentration and no degradation reduction was observed even at the highest initial concentration (400 μM) ( Supplementary Fig. S6). Thus, we confirmed that our chitinase enzymatic assay was not affected by transglycosylation. The peak activity was observed at pH 5.0 with high levels between pH 1.0-6.0 and being present even at pH 7.0 (Fig. 2a). When the Mf-CHIA activity level at pH 5.0 was set to 100%, the relative activity at pH 2.0 (Gly-HCl buffer), pH 2.0 (McIlvaine's buffer) and pH 7.0 (McIlvaine's buffer) were 62%, 52% and 31%, respectively. Notably, the recombinant Mf-CHIA had properties very similar to the native enzyme from stomach extracts of the crab-eating monkey for the pH preference 29 .
When compared to Mm-CHIA, Mf-CHIA had higher activity at each condition. There was a threefold difference in their peak activities Mf-CHIA at pH 5.0, Mm-CHIA at pH 2.0 being 182.1 U/μg and 57.83 U/μg protein, respectively (Fig. 2b). In addition, Mf-CHIA was also 2×, 16×, and 10× more active at pH 2.0, 5.0, and 7.0, respectively, than the mouse enzyme. Thus, Mf-CHIA's chitinolytic activity properties differ from other animals reported previously [30][31][32][33]35,36 and are rather similar to human CHIA, at least in regard to pH 23,34,37 . Temperature dependence of Mf-CHIA activity. The effect of temperature on enzyme activity was determined in McIlvaine's buffer at pH 2.0, 5.0 or 7.0 and 30-70 °C for 60 min. Figure 3 shows relative activities of both enzymes where the peak activity of Mf-CHIA under optimal conditions (pH 5.0 and 65 °C) was set to 100%. At pH 2.0, the optimal value for Mm-CHIA, Mf-CHIA was most active at 55 ℃ (Fig. 3a). As shown in Fig. 3b (pH 5.0), the reaction rate increased with the temperature and reached the maximum level at 65 °C. At pH 7.0, Mf-CHIA reached the peak activity at 37 ℃ and then rapidly decreased with increasing temperature (Fig. 3c). More detailed comparison of the temperature-dependence between Mf-CHIA and Mm-CHIA is shown in Supplementary Fig. S7. At pH 2.0, Mf-CHIA retained high activity at 65 ℃, whereas in Mm-CHIA, the activity markedly dropped at > 60 ℃. At pH 5.0, the monkey enzyme maintained its high activity even at 70 ℃ (approximately 80%). At pH 7.0, the activity of Mf-CHIA dropped at > 50 ℃, on the other hand, Mm-CHIA retained its (relatively low) activity at 50 ℃.
Overall, chitinolytic activity of Mf-CHIA significantly exceeded that of Mm-CHIA at each tested condition (Fig. 3). while demonstrating high thermostability.
pH stability of the Mf-CHIA. Next, we aimed to determine the pH stability of the enzyme at different temperatures. Mf-CHIA was pre-incubated at 0, 37 or 65 ℃ for 60 min at pH 1.0-8.0. Pre-incubation was followed by enzymatic activity analysis at 37 °C and pH 5.0 for 60 min. We show the relative activity when the highest residual activity of Mf-CHIA was set to 100%. As shown in Fig. 4a, Mf-CHIA remained stable over a broad pH range (pH 1.0-8.0), during the 1-h pre-incubation at 0 ℃. This treatment caused no measurable decrease in chitinase activity. Thermal stability of the Mf-CHIA. We assessed the Mf-CHIA's thermal stability with pre-incubation of the samples at pH 2.0, 5.0 or 7.0 for 30 min at 30-80 °C. The residual activity was measured using the chromogenic substrate at 37 °C and pH 5.0 for 60 min. We show the relative activity when the highest residual activity of Mf-CHIA was set to 100%. Pre-incubation at pH 2.0 resulted in the enzyme's deactivation above 60 ℃, where it retained 60% of its peak activity (Fig. 5a). The enzyme also remained stable to up to 70 ℃ at pH 5.0 and at 80 ℃, there was still 31% activity present (Fig. 5b). At pH 7.0, Mf-CHIA was stable to up to 40 ℃ (Fig. 5c). Thus, Mf-CHIA is stable at 30-70 ℃ depending on the pH conditions.  38,39 . We quantified the (GlcNAc) 2 and (GlcNAc) 3 produced by the enzymes under each pH and temperature condition ( Fig. 6 and Supplementary Figs. S8 and S9). We show the relative activity when the Mf-CHIA degradation product's peak was set to 100%. At 37 ℃ and 50 ℃, high levels of (GlcNAc) 2 produced by Mf-CHIA were observed at pH 2.0 and pH 5.0 (Fig. 6a,b,    www.nature.com/scientificreports/ tion products with Mf-CHIA were obtained at pH 2.0, although the optimal pH level was 5.0. On the other hand, at 65 ℃, the optimal pH was 5.0 where both dimer and trimer production peaked (Fig. 6c,f 3 was also produced by both Mf-CHIA and Mm-CHIA, although it did not reach the amount of (GlcNAc) 2 . Under each condition, Mf-CHIA performed more efficiently than Mm-CHIA.
Mf-CHIA was three times more active than Mm-CHIA under respective optimal pH levels ( Fig. 2a) and significantly higher at all tested conditions (Figs. 2 and 3). Expression and activity levels of acidic chitinases are much higher in omnivorous animals in comparison with carnivorous and herbivorous animals 36 . It has been reported that Mf-CHIA is 50 times more active than human CHIA 37 . However, pH-dependent profiles of Mf-CHIA were similar to those of human CHIA 23,34,37 .
Mf-CHIA was most active at 65 ℃ and pH 5.0 (optimal pH) and more efficient in artificial chromogenic (Figs. 2 and 3) and polymeric chitin substrates (Fig. 6) degradation at temperatures (50-70 ℃) above normal body temperature (37 ℃). Moreover, the remarkable stability of this enzyme was demonstrated also in strong acidic environment (Figs. 4a,b, 5a). Thus, we clarified the enzymatic properties of Mf-CHIA and identified the inactivating conditions.
Using polymeric chitin, Mf-CHIA produced at 37 ℃ and 50 ℃ more degradation products at pH 2.0 than at its optimal pH 5.0 (Fig. 6). The active center (DXXDXDXE motif) in Chia proteins, including Mf-CHIA, is thought to have an essential role in substrate binding and catalysis in acidic conditions, with His187 being responsible for the acidic optimum 41 . Interestingly, the optimal condition for polymeric chitin degradation (pH 2.0, 50 ℃) differs from the optimal condition for chromogenic substrate degradation by CHIA (pH 5.0, 65 ℃). The reason for this discrepancy is yet to be revealed and is under further investigation.
In this study, we show a detailed characterization of Macaca fascicularis CHIA (Mf-CHIA). Mf-CHIA seems to be more active toward 4-NP-(GlcNAc) 2 chromogenic substrate than toward polymeric chitin (Figs. 2, 3 and  6). This is in agreement with a previous report showing that activity variability between experiments using chromogenic substrate and polymeric chitin may be driven by differential substrate specificity 12 .
Chitooligosaccharides have been reported to have various anti-tumor and anti-inflammatory activity while being involved in certain metabolic diseases [42][43][44][45] . Mf-CHIA activity was high under a broad range of pH and temperature conditions, demonstrating its acid-and thermostability. Our present results also indicate that Mf-CHIA has a promising potential to become the enzyme of choice for chitooligosaccharides production for agricultural and medical purposes.

Methods
Monkey and mouse total RNAs. The study was designed and carried out in compliance with the ARRIVE guidelines 46 . We purchased crab-eating monkey (Macaca fascicularis) total RNA from UNITECH Co., Ltd., Chiba, Japan, and mouse total RNAs (BALB/c mice) from and Takara Bio USA, Inc., Mountain View, CA, USA. We did not use living animals but expressed proteins in E. coli. The use of animal-derived total RNAs and all procedures in this study were reviewed and approved by the Recombinant DNA Committee at Kogakuin University.

E. coli expression vectors.
We used monkey or mouse stomach total RNAs and reverse transcribed as previously described 29,33 . Coding regions of the mature form of Macaca fascicularis CHIA (Mf-CHIA) and Mus musculus CHIA (Mm-CHIA) cDNAs were amplified from the corresponding animal's cDNAs by PCR using KOD Plus DNA polymerase (Toyobo Co., Ltd, Osaka, Japan) and oligonucleotide primers (Eurofins Genomics, Tokyo, Japan) anchored with the restriction sites for BamHI or XhoI (Supplementary Table S1) as described previously 33 . We obtained monkey and mouse cDNA by reverse transcription of total RNA. Amplified cDNA was digested with BamHI and XhoI and inserted into the pEZZ18 vector. The entire nucleotide sequence of the resulting plasmid DNA (pEZZ18/CHIA/V5-His) was confirmed by sequencing (Eurofins Genomics). Expression of these plasmid DNA in E. coli cells led to the production of the mature Protein A-CHIA-V5-His.
Preparation of the recombinant chitinase proteins expressed in E. coli. E. coli BL21 (DE3) (Merck Millipore, Tokyo, Japan) was transformed by pEZZ18/pre-Protein A-CHIA-V5-His to express pre-Protein A-Mf-CHIA-V5-His or pre-Protein A-Mm-CHIA-V5-His proteins. Transformed E. coli BL21 (DE3) strains were grown in 3 L LB medium containing 100 µg/mL ampicillin at 37 °C for 18 h. Cells were harvested by centrifugation at 7,000g for 20 min at 4 °C. The recombinant protein in the soluble fraction was passed through the IgG Sepharose column (GE Healthcare, Piscataway, NJ, USA) as described previously 33 . The protein-containing fractions were desalted using PD MidiTrap G-25 (GE Healthcare) equilibrated with the TS buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl and a protease inhibitor (Complete, Roche, Basel, Switzerland)]. We analyzed the protein fractions using standard SDS-PAGE, followed by Western blot. Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Merck Millipore), which was probed using a polyclonal anti-V5-HRP monoclonal antibody (Invitrogen, Carlsbad, CA, USA). We also conducted SYPRO Ruby staining (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer`s instructions. We analyzed and quantified the immunoblots using the Luminescent Image Analyzer (ImageQuant LAS 4000, GE Health- Zymography assays. We performed zymography analysis using standard SDS-PAGE gel except for containing 0.1% ethylene glycol chitin (Wako Pure Chemical Industries). Samples were loaded without heat denaturation in SDS-free sample buffer. After electrophoresis, we stained gel using Calcofluor white M2R (Sigma-Aldrich) as described previously 30 . The gels were analyzed using the Luminescent Image Analyzer.
Chitinase enzymatic assays. We determined chitinolytic activity using 4-nitrophenyl N,N′-diacetyl-β-D- The degradation products were labeled and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), as described previously 38,39 . We took all gels with the same exposures. N-acetyl chitooligosaccharides (Seikagaku Corporation, Tokyo, Japan) were used as a standard.
Statistical analysis. Biochemical data were compared by Student's t-test.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.