Purification of chitosanase from Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 for chitooligosacharide production

Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 are bacterial strains isolated from shrimp waste capable of hydrolyzing chitosan to produce chitooligosaccharides (COSs). Their COS products showed antimicrobial activity. The aim of this study was to purify chitosanase from those bacteria and used for COSs production. Chitosanase from both strains were produced using 0.5% colloidal chitin as inducer. The chitosanase was then purified by ultrafiltration, DEAE Sepharose ion exchange and Separacyl S-300 gel filtration chromatography. The specific activity and the yield of chitosanase KPU 2123 increased 2.35-fold and 30.90% after three steps of purifications, respectively, whereas chitosanase T5a1 increased 3.32-fold and 22.79%, respectively. The molecular weight of both chitosanases KPU 2123 and T5a1 were about 76 and 20 kDa, respectively. The COS products generated by chitosanase KPU 2123 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine, Tri-N-acetyl-D-glucosamine, Tetra-acetyl-D-glucosamine and Penta-N-acetyl-D-glucosamine while by chitosanase T5a1 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine. Based on their COS products, chitosanase KPU2123 can be categorized as endo-type chitosanase. Further study is needed to analysis the bioactivity of COSs obtained from the pure of chitosanases KPU 2123 and T5a1.


Introduction
Chitin and chitosan have been developed and used in various industries as biomaterials for food, pharmaceutical, textile, and other sectors (Choi et al 2004). Their derivative products, chitin oligosaccharides (CTOSs) and chitosan oligosaccharides (COSs) are known more valuable than their original material because of their biological activities (Wang et al 2008). Transforming chitosan to COS increases physiochemical properties such as water solubility and nontoxicity (Lodhi et al 2014). Furthermore COSs were reported having biological properties for decreasing cholesterol, enhancing calcium absorption, and improving antihypertensive, antioxidant, antimicrobial and antitumor effects (Aam et al 2010, Kim 2012, Yang et al 2019). Conventionally, COSs are produced by chemical processes. This method however, produced a small number of oligosaccharides, which contained short chain of glucosamine units, and secondary compounds that are difficult to be separated in the purification process (Mourya et al 2011, Wang et al 2008. Acid or alkaline hydrolysis is also considered not environment-friendly because of its waste pollution (Choi et al 2002). Furthermore, the chemical residues make COS not applicable for human consumption (Je and Kim 2012). An alternative to replace chemical method is enzymatic process, which is eco-friendly, easy to control and generate COSs without any harmful side molecules (Liang et al 2018, Su et al 2006. Chitosanase is one of the enzymes that hydrolyze chitosan to COSs (Lodhi et al 2014).
Chitosanases (EC 3.2.1.132) are enzymes catalyzing hydrolysis of β 1-4 glycosidic bond of the chitosan releasing mixture of N-glucosamine (Rodriguez-Herrera et al 2008, Pechsrichuang et al 2018. These enzymes are mostly found in microorganisms, including bacteria and fungi (Choi et al 2004, Wang et al 2008. Chitosanases are classified into seven families based on their action of glycoside hydrolase (GH). The mechanism actions of chitosanases are different depending on their sources. Bacteria chitosanases are often grouped in GH family 46, while fungi chitosanases are in family 75 (Qin et al 2018). The isolation and development of chitosanases have been attracting researchers and industry, especially cold adaptive and thermostable chitosanases (Qin et al 2018, Zhou et al 2019.
In the previous studies, our group has isolated chitinolytic bacteria from marine environments and shrimp waste. A total of 106 isolates were found including Stenotrophomonas maltophilia KPU 2123 and . The production of those COSs were conducted using crude enzyme. Therefore, in this study, we reported the purification of chitosanases from Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1, and production of COSs using the purified enzymes.

Materials
The strains of Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 were a collection of Biotechnology laboratory at RCMFPPB, isolated from shrimp waste and shrimp paste, respectively (Chasanah et al 2007, . Chitosan was obtained from Bogor Agricultural University (IPB University). COSs standard (1-6 units) was purchased from Seikagaku Corp., Japan, while glucosamine from Sigma. Other chemicals and microbiological media were in analytical grade.

Purification of chitosanase.
The chitosanase purification consisted of three stages. The supernatants were firstly subjected to ultrafiltration using membrane with MWCO 10 kDa (GE Healthcare) until reached concentration of 10 times. The concentrated enzymes were applied into ion exchange chromatography using DEAE Sepharose TM Fast Flow (GE Healthcare) column in AKTA Purifier (GE Healthcare). The enzymes were eluted using gradient concentration of 0.02 M Tris-Cl buffer pH 9 and 1 M NaCl. The active fractions were further purified using HiPrep Separacyl S-300 (GE Healthcare) in the gel filtration chromatography system. As a mobile phase, 0.02 M of Tris-Cl buffer pH 9 was used. The chitosanase activity and total soluble protein of each purification steps were assayed. The purity of each step was analyzed using 10% SDS-PAGE. 2.2.3. Chitosanase activity. The activity of chitosanase was determined based on Schales method as reported by Yoon et al (2000). Soluble chitosan 1% was used as a substrate prepared according to Choi et al (2004), while glucosamine was used as a standard.
2.2.4. Protein content. The protein content of the enzyme was measured using Lowry method (Bollag and Edelstein 1991). A serial concentration of bovine serum albumin (BSA) from 0 to 0.2 mg/mL was used as a standard.
2.2.5. Production of chitooligosaccharides. Chitooligosaccharides were produced using chitosanase at a concentration of 8 U/gram of chitosan. The mixture was incubated for 16 hours. The reaction was inactivated by boiling the mixture for 10 minutes. The mixture then was centrifuged at 9,000 g for 15 minutes to separate the COSs and the residue. The COSs content was analyzed using HPLC. A Shodex Asahipak NH2P-50 coloumn was used. The COSs were eluted using a mixture of acetonitrile-water (70:30) at flow rate 1 mL/minute. A mixture of monomer to hexamer chitooligosaccharides was used as a standard (Seikagaku Corp., Japan).

Results and discussion
Purification results of chitosanases are summarized in table 1. Ultrafiltration increased the specific activity of chitosanase KPU 2123 from 0.83 to 1.46 U/mg (1.76 fold), and specific activity of chitosanase T5a1 from 2.55 to 2.99 U/mg (1.17-fold). In addition, this process produced enzymes with 76.48 and 46.94% of yield respectively. Ultrafiltration with 10 kDa cut off membrane will hold protein above 10 kDa in the reservoir and remove the smaller ones (Fawzya et al 2018).   1a and 1b). Thus, a further purification step is required. This result was similar to the purification of chitosanase from Serratia marcescens, which required gel filtration process after DEAE ion exchange (Wang et al 2008). DEAE ion exchange is categorized as anion exchange chromatography resin. This resin will bind the negative charge of proteins (Scopes 2013). The sepacryl S-300 purification profiles of KPU2123 and T5a1 chitosanases are shown in figure 3. The KPU2123 had three peaks in its profile, while the T5a1 had five peaks. Although the number of peaks for both enzymes were different, the first three peaks had similarity in term of their elution. SDS PAGE results showed that only peak number two for both KPU 2123 and T5a1 had chitosanase activity ( figure 4). The size of the bands was about 76 kDa and 20 kDa for KPU2123 and T5a1 respectively. After this Sepacryl purification step, the specific activity of KPU2123 was 1.95 U/mg, 2.35-fold greater than that of the crude enzyme, with the yield was 30.90%. Meanwhile, the specific activity of T5a1 was greater (8.45 U/mg) than that of KPU 2123. This activity increased 3.32-fold from the initial activity. The enzyme yield of T5a1 was 22.79% (table 1)   depending on the type of enzymes. The molecular weight of chitosanase ChiA from Aspergillus sp. was reported 109 kDa, while ChiB from the same sources was 29 kDa. The size of other chitosanases from Aspergillus spp. was between 22.5 kDa to 135 kDa where endo-type chitosanase (22.5-40 kDa) has lower molecular weight compared to the exo-type chitosanase (108-135 kDa) (Chen et al 2005). The molecular weight of chitosanase from T5a1 was similar to that of chitosahase from Serratia marcescens (21 kDa) and chitosanase C from Bachillus megaterium (22 kDa) (Pelletier and Sygusch 1990). Whereas the size of chitosanase from KPU2123 was different from other chitosanases that have been reported. The nearest sizes were chitosanases from Serratia plymuthica with molecular weight of 60.5 and 95.6 kDa (Wang et al 2008).