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Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein
Protein Expression and Purification 166 (2020) 105519
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein
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Yu Liua,1, Yanling Lib,1, Sheng Tongb, Min Yuanb, Xiaoyun Wangb, Junyao Wangb, Yanhua Fanb,∗ a b
College of Biotechnology, Southwest University, 400716, Beibei, Chongqing, PR China Biotechnology Research Center, Southwest University, 400716, Beibei, Chongqing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Beauveria bassiana Chitosanase Pichia pastoris Purification Enzymatic characteristics
Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 μg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2–8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.
1. Introduction Chitosanase (EC 3.2.1.132) can specifically catalyze the breakdown of the β-1,4-glucoside bonds in chitosan and release chitooligosaccharides (COSs) or glucosamine [1]. The first studied chitosanase was of microbial origin and can degrade Zygomycete cell walls [2]. Over the past 40 years, chitosanases have been found in different microorganisms and have shown potential for COS preparation and antifungal applications with their ability to degrade fungal cell walls [3–6]. The degrading products of chitosan by chitosanase, COSs, are alkaline oligosaccharides with multiple biological activities, such as anticancer, antioxidant, and immunostimulant effects. In addition, COSs also exhibit antifungal activity, which can antagonize plant pathogens and induce plant defense reactions [7,8]. Therefore, COSs have potential applications in medicine, agriculture, food, cosmetics and other fields and have attracted increasing attention. However, presently, only a limited number of chitosanases have been characterized, and enzymes from various sources have exhibited different enzymatic properties. To explore the application of chitosanase, it is necessary to characterize various chitosanases from different sources. Beauveria bassiana, an entomopathogenic fungus, has been widely used for the biocontrol of agricultural, forest, and urban pests [9]. In addition to inhabiting infected insect hosts, B. bassiana has other lifestyles, including saprophytes and endophytes [10]. Some hydrolases
have been found in this fungus, such as chitinases, proteases, and lipases [11]. These hydrolases have been verified to play vital roles in the fungal infection process against insect hosts by degrading host cuticles or being involved in antagonistic processes against phytopathogenic fungi [12,13]. Similar to chitinase and protease, B. bassiana chitosanase may also be involved in fungal pathogenesis or have antagonistic activity against other fungi. However, no chitosanase from entomopathogenic fungi has been characterized. In the genome of B. bassiana, there is a chitosanase-encoding gene (named BbCSN-1) that encodes a protein of 300 amino acids. To characterize BbCSN-1, the protein was heterologously expressed in the methylotrophic yeast Pichia pastoris, which can modify proteins by glycosylation, spatial folding, protease processing and the formation of disulfide bonds [14]. The expressed BbCSN-1 was purified from the culture supernatant, and the enzymatic properties were characterized with colloidal chitosan. 2. Materials and methods 2.1. Chemicals T4 DNA ligase was purchased from Promega (USA). All restriction endonucleases were from MBI (USA). The prestained protein marker was from Thermo (USA). The PCR primer was prepared by YingJun Biotechnology Company (Shanghai, China). Yeast extract and peptone
Corresponding author. E-mail address: [email protected] (Y. Fan). 1 Yu liu and Yanling Li contributed equally to this work. ∗
https://doi.org/10.1016/j.pep.2019.105519 Received 24 May 2019; Received in revised form 16 October 2019; Accepted 16 October 2019 Available online 18 October 2019 1046-5928/ © 2019 Published by Elsevier Inc.
Protein Expression and Purification 166 (2020) 105519
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Fig. 1. Amino acid sequence and phylogenetic tree of BbCSN-1. (A) Amino acid sequence of BbCSN-1. (B) Phylogenetic tree of BbCSN-1 showing homologous proteins. (C) Construction map of recombinant expression vector pPIC9K-BbCSN-1 in P. pastoris.
2
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were from Sigma (USA). The DNA agarose recycling kit was from BioFlux (USA). PDA (potato dextrose agar) and PDB (potato dextrose broth) media were from BD (USA). The plasmid extraction kit was from Omega (USA). D-(+)-Glucosamine was purchased from Solar Biotechnology Co., Ltd. (Beijing, China). All chemicals used were of analytical grade. The protein deglycosylation Mix Ⅱ (P6044) was from BioLabs. (New England).
Superdex™ 75 pg, GE, USA). The column was equilibrated with 20 mM sodium acetate buffer (pH 5.4), and the protein was eluted with the same buffer at a flow rate of 0.7 ml/min. The purified protein was dialyzed against 20 mM sodium phosphate buffer (pH 5.0). N-terminal sequence of the purified BbCSN-1 was determined by Protein Sequencer PPSQ-31A (Shimadzu Corporation, Japan). Protein purity was analyzed based on densitometry (Bandscan soft).
2.2. Plasmids and strains
2.7. Enzymatic activity analysis of BbCSN-1
The A-T clone vectors pGEM-T and pEASY-Blunt were products from Promega and TransGen Biotech, respectively. The plasmid pPIC9K and P. pastoris GS115 strain were purchased from Invitrogen (USA). DH5α (E. coli) was purchased from TaKaRa (China).
To prepare colloidal chitosan, 1 g of chitosan powder was dissolved in 100 ml of 1% HCl. The pH of the chitosan solution was adjusted to 5.0 with NaOH. One milliliter of colloidal chitosan was collected by centrifugation (10,000 rpm, 5 min) in a 1.5 ml Eppendorf tube and washed with the same volume of Tris-HCl solution (1 M, pH 8.0) three times. Then, the substrate was resuspended in 1 ml of potassium phosphate buffer (20 mM, pH 5.0) and used for chitosanase analysis. The reaction mixture contained 100 μl of colloidal chitosan substrate, 100 μl of BbCSN-1, and 150 μl of potassium phosphate buffer (20 mM, pH 5.0). After reacting for 1 h at 30 °C, the reaction mixture was centrifuged, and 200 μl of supernatant was transferred into a new 1.5 ml Eppendorf tube, and 200 μl of DNS (3,5-dinitrosalicylic acid) and 100 μl of 1 M NaOH were added. After placing the tube at 95 °C for 10 min, the absorption value (OD540) was measured. D-Glucosamine was used to prepare a standard curve. One unit of chitosanase activity was defined as the amount of enzyme required to release 1 μM of D-glucosamine per hour. Three parallel groups were set up for each experimental group.
2.3. Expression plasmid construction of BbCSN-1 The intact coding region of BbCSN-1 in B. bassiana was obtained by PCR with primers Bbcsn-ye1 and Bbcsn-ye2 (Bbcsn-ye1: 5′-gaattccgcgacgtaccgtccaacgt-3′, Bbcsn-ye2: 5′-gcggccgcttaaccacgggcacacttgc-3’; the underline sequences are EcoRI and NotI sites, respectively) using B. bassiana genomic DNA as a template. There is no intron in this gene. The PCR product was cloned into the pGEM-T vector for DNA sequencing. The BbCSN-1 gene was excised from the pGEM-T vector with EcoRI, and the NotI site was used to insert the BbCSN-1 gene into the corresponding sites of the pPIC9K vector to produce pPIC9K-BbCSN-1. The recombinant fragment was transformed into E. coli DH5α, and the correct vector was verified by sequencing.
2.8. Characteristics of recombinant BbCSN-1
2.4. Transformation and selection of positive transformants
To determine the optimal reaction temperature, chitosanase activity was measured in potassium phosphate buffer (20 mM pH 5.0) at different temperatures (10, 20, 30, 40, 50 and 60 °C). To determine the optimal pH, the purified chitosanase activity was measured at different pH values (ranging from pH 2.0 to pH 10.0: pH 2.0–3.0, achieved with 0.05 M glycine-HCl buffer; pH 4.0–5.0, achieved with 0.2 M CH3COONa–CH3COOH buffer; pH 6.0–8.0, achieved with 0.1 M potassium phosphate buffer; and pH 9.0–10.0, achieved with 0.05 M glycine-NaOH buffer) for 30 min at 30 °C. The temperature and pH stability of BbCSN-1 were analyzed by using colloidal chitosan as a substrate. BbCSN-1 was pretreated for 30 min under different temperatures (10, 20, 30, 40, 50 and 60 °C) or different pH buffers as described above. Then, the chitosanase activity was measured with colloidal chitosan at pH 5.0 and 30 °C. To determine the optimal pH, the purified Bbchit1 activity was measured at different pHs (ranged from pH 3.0 to pH 10.0: pH 3.0-pH 6.0 was 0.1 M citrate–trisodium citrate buffer; pH 7.0–8.0 was 0.2 M potassium phosphate buffer; pH 9.0–10.0 was 0.05 M glycine–sodium hydroxide buffer) at 37 °C for 1 h. To determine the optimal reaction temperature, chitinase activity was measured in 0.2 M potassium phosphate buffer, pH 6.4, at different temperatures, 4, 15, 25, 35, 45, 55, and 65 °C, respectively.
pPIC9K-BbCSN-1 plasmid DNA was isolated and linearized with PmeI. The digested DNA was transformed into the yeast strain P. pastoris GS115 by electroporation according to the operating manual. In order to obtain yeast colonies with multiple copies of BbCSN-1, fast-growing positive transformants were selected in yeast peptone dextrose adenine (YPDA) medium supplemented with the antibiotic G418 (1.75 mg/ml) and confirmed by PCR. 2.5. Fermentation cultivation of BbCSN-1 in P. pastoris A colony of P. pastoris expressing BbCSN-1 was inoculated into 150 ml of buffered glycerol-complex medium (BMGY) (1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base with ammonium sulfate and without amino acids, 4 × 10−5% biotin, 100 mM potassium phosphate, pH 6.0, and 1.0% glycerol) grown at 28 °C in a shaker incubator. The cells were harvested by centrifugation and resuspended in 100 ml of buffered methanol-complex medium (BMMY) (1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base with ammonium sulfate and without amino acids, 4 × 10−5% biotin, 100 mM potassium phosphate, pH 6.0, and 1% methanol). The suspension was transferred to a sterile 1-L baffled flask and grown at 28 °C. To determine the time course of BbCSN-1 expression in P. pastoris, the fermented supernatant was collected daily (0–7 d) and analyzed for the expression of BbCSN-1 (SDS-PAGE), total protein concentration (Bradford method), activity of chitosanase and other experiments.
2.9. The influence of metal ions and EDTA on BbCSN-1 activity To evaluate the effects of metal ions on chitosanase activity, metal ions (5 mM), including MnCl2, FeCl3, CaCl2, BaCl2, CoCl2, MgCl2, CuSO4, FeSO4, KCl, NaCl, LiCl and EDTA were added to the reaction mixture. The relative activity was measured as the ratio between the chitosanase activity with metal ions and that without metal ions [15].
2.6. Purification recombinant BbCSN-1 from P. pastoris For purification of the recombinant BbCSN-1 from P. pastoris, the culture supernatant was concentrated by PEG 20,000 in a bag filter (MWCO 14000). The concentrated supernatant was directly subjected to desalting chromatography (AKTAprime plus, HiPrep™ 26/10 desalting chromatography column, GE, USA) equilibrated with 50 mM sodium acetate buffer (pH 5.4). The fractions containing BbCSN-1 were further purified by molecular sieve chromatography (HiLoad™ 16/600
2.10. The enzymatic kinetics of BbCSN-1 Colloidal chitosan (1–5 mg/ml) was used as a substrate to measure enzyme kinetics under standard conditions (20 mM potassium phosphate buffer, pH 5.0; 30 °C). Km and Vmax of BbCSN-1 were calculated 3
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Fig. 2. P. pastoris transformants of BbCSN-1 verified by PCR. Lane M, marker 2000; Lane 1, sterile water; Lane 2, pPIC9K plasmid; Lane 3, pPIC9KBbCSN-1 plasmid; Lane 4–6, different BbCSN-1 colonies. The arrow indicates the BbCSN-1 fragment.
by the Lineweaver-Burk double inverse method. 2.11. Analysis of chitosan oligosaccharides To understand the degradation pattern of BbCSN-1 against chitosan oligosaccharides, the TLC method was conducted as described by Choi [16]. 2.12. Enzymatic deglycosylation and disulphide bonds analysis of BbCSN-1
Fig. 4. Purified BbCSN-1 from P. pastoris culture supernatant. (A) Profile of BbCSN-1 purified by desalting chromatography. Peak 1 contained BbCSN-1 protein and chitosanase activity was detected (88.06U/mg). (B) Profile of BbCSN-1 purified by a molecular sieve. The fractions of peak 1–4 were collected and analyzed by SDS-PAGE. Activity of each peak was detected and only Peak 2 showed chitosanase activity (101.11U/mg).
BbCSN-1 was treated by enzymatic deglycosylation to determine whether this protein is glycosylated. Reaction protocols were followed by manufacturer's instructions. Control protein and BbCSN-1 were detected by SDS-PAGE respectively. To determine whether there are disulphide bonds in recombinant BbCSN-1, the recombinant protein was subjected for SDS-PAGE analysis under reducing (with 12% β-mercaptoethanol in loading buffer) and non-reducing conditions (without β-mercaptoethanol).
heterologously expressed in P. pastoris GS115. The expression vector pPIC9K-BbCSN-1 was constructed following the procedure described in the Materials and Methods (Fig. 1C). The obtained expression vector was transformed into P. pastoris cells by electroporation and grew on YPD plates containing 1.75 mg/ml G418 for screening multi-copy colony. Three rapidly growing positive transformants were selected and verified by PCR (Fig. 2).
3. Results and discussion 3.1. BbCSN-1 expression plasmid construction and selection of BbCSN-1 P. pastoris positive transformants The B. bassiana chitosanase gene BbCSN-1 encodes a protein of 300 amino acids (accession number: EJP64701) with an 18-aa N-terminal signal peptide predicted by Signal P. BbCSN-1 showed 99% identity to the homologous proteins from Cordyceps brongniartii (Fig. 1A and B). To analyze the enzymatic characteristics of BbCSN-1, BbCSN-1 was
3.2. Expression of BbCSN-1 in P. pastoris Three P. pastoris GS115 colonies containing BbCSN-1 were cultured in BMGY/BMMY culture medium and induced with 0.5% methanol to produce BbCSN-1. The supernatant of cultured P. pastoris cells was
Fig. 3. Time-course changes of BbCSN-1 expression in P. pastoris. (A) The expression of BbCSN-1 induced for different times was detected by 12% SDS-PAGE. Lane M, molecular weight marker; Lane P, fermentation broth of P. pastoris transformed with pPIC9K induced by methanol; Lane 1–7, fermentation broth of P. pastoris transformed with pPIC9K-BbCSN-1 induced by methanol for one day to seven days. Twenty microliters of pretreated sample was loaded in every lane. (B) The expression of BbCSN-1 on different induction days was measured by the Bradford method. 4
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detected daily by SDS-PAGE for the production of BbCSN-1. As the tested three colonies exhibited similar expression level of BbCSN-1 (data not shown), only one colony's results were shown here. Compared to the wild-type strain, a protein with a molecular mass of approximately 33 kDa, which was similar to the theoretical value of BbCSN-1 (Fig. 3A). The results showed that BbCSN-1 was expressed from the first day of induction with a protein amount of 60 μg/ml. The amount of BbCSN-1 gradually increased within 1–6 d and reached a peak at 398 μg/ml on the 6th day (Fig. 3B). These results indicated that BbCSN1 could be successfully expressed in P. pastoris GS115. The N-terminal sequence of the recombinant BbCSN-1 is EAEAYE, corresponding to the predicted cleave site of protease KEX2 signal cleavage. 3.3. Purification of recombinant BbCSN-1 from P. pastoris Fig. 5. SDS–PAGE analysis of BbCSN-1 after purification. Lane M, molecular mass standards indicated in kDa; Lane 1, blank vector sample; Lane 2, fraction after desalting chromatography; Lanes 3–6, peak from samples 1–4 after molecular sieve chromatography as shown in Fig. 4B.
To purify recombinant BbCSN-1 from P. pastoris, the supernatant was concentrated by 5-fold in a dialysis bag (MWCO 14 kDa). The sample was subjected to desalting chromatography (Fig. 4A). The fraction with chitosanase activity (Peak 1 in Fig. 4B) was further purified by molecular sieve chromatography (Fig. 4B, Fig. 5). After
Table 1 Purification of the BbCSN-1 from Beauveria bassiana expressed in P. pastoris. Purification steps
Total activity (U/L)
Total protein (mg/L)
Specific activity (U/mg)
Purification factor
Yield (%)
Culture supernatant Desalting Molecular sieve chromatography
7518.85 4100.71 2586.63
1371.74 46.57 25.58
5.48 88.06 101.11
1.00 16.07 18.45
100.00 54.54 34.40
Fig. 6. Effects of temperature and pH on chitosanase activity. (A) Effect of temperature. The chitosanase activity of BbCSN-1 was measured at temperatures ranging from 4 °C to 60 °C at pH 5.0 for 30 min. (B) Temperature stability of BbCSN-1. After BbCSN-1 was pretreated at temperatures ranging from 10 °C to 60 °C at pH 5.0 for 30 min, chitosanase activity was analyzed under standard conditions (pH 5.0, 30 °C). (C) Effect of pH. The chitosanase activity of BbCSN-1 was determined under a pH range from pH 2.0 to 10.0 at 30 °C for 30 min. The buffers used were 0.05 M glycine-HCl buffer (pH 2.0–3.0), 0.2 M sodium acetate-acetic acid buffer (pH 4.0–5.0), 0.1 M potassium phosphate buffer (pH 6.0–8.0) and 0.05 M glycine-NaOH buffer (pH 9.0–10.0). (D) pH stability of BbCSN-1. After BbCSN-1 was treated at temperatures ranging from pH 2.0–10.0 at 30 °C for 30 min, chitosanase activity was analyzed under standard conditions (pH 5.0, 30 °C). 5
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3.5. Enzyme kinetic parameters of purified BbCSN-1
Table 2 Effect of various metal ions and EDTA on chitosanase activity. Chemicals
Relative activity (%)
Chemicals
Relative activity (%)
None Mn2+ Fe3+ Na+ K+ Cu2+ Mg2+
100 233.3 ± 1.0 99.3 ± 1.2 118.0 ± 2.6 105.1 ± 5.5 51.9 ± 2.5 111.8 ± 4.4
Li+ Fe2+ Co2+ Ba2+ Ca2+ EDTA
102.9 ± 5.0 106.0 ± 2.8 50.9 ± 0.5 121.4 ± 1.5 99.2 ± 1.0 82.3 ± 4.0
To study the affinity between BbCSN-1 and the chitosan substrate, the Km of BbCSN-1 was detected. Based on the Lineweaver-Burk plotting method, the Km and Vmax of BbCSN-1 were approximately 1.52 mg/ml and 5.0 μg/min at 30 °C (pH 6.0), respectively. 3.6. Reaction pattern of purified BbCSN-1 The reaction products of purified BbCSN-1 were examined by TLC using several chitosan oligosaccharides as the substrates. The results
5 mM metal ions and EDTA were added to the enzymatic reaction mixture and chitosanase activity was measured with colloid chitosan as substrate (20 mM potassium phosphate buffer pH 5.0, 30 °C). The relative activity was the ratio of chitosanase activity with metal ions to the chitosanse without metal ion under the same reaction conditions.
purification, chitosanase in Peak 2 was obtained and protein purity is 80.4%. The specific activity of purified BbCSN-1 was determined to be 101.11 U/mg with an 18-fold increase compared to the original supernatant. The overall yield was estimated at 34.40%. (Table 1). 3.4. Enzymatic properties of purified BbCSN-1 The enzymatic properties of BbCSN-1 were measured with colloidal chitosan as a substrate [17,18]. The purified BbCSN-1 exhibited optimum activity at 30 °C and pH 5 (Fig. 6A, Fig. 6C). The stability analysis showed that BbCSN-1 was stable below 40 °C and that the relative activity was over 80% (Fig. 6B). However, the relative activity decreased to 60% after treatment at 50 °C or 60 °C for 30 min. In addition, BbCSN-1 was stable when the pH was 2–5; however, the relative activity gradually decreased with increasing pH (pH > 5) (Fig. 6D). The high pH conditions may cause the degradation of chitosanase or decrease chitosan substrate solubility, resulting in the decrease in enzyme activity. The influence of metal ions (5 mM) on the activity of BbCSN-1 was determined (Table 2). Mn2+ had a significant activation effect on the activity of BbCSN-1 and enhanced the activity of BbCSN-1 by 133%. However, Co2+ and Cu2+ had a strong inhibitory effect on the activity of BbCSN-1. With 5 mM Co2+ and Cu2+, the relative activity of BbCSN1 was decreased by 50%. Fe3+, Li+ and Ca2+ had no significant effect on the activity of BbCSN-1. Other metal ions, such as Na+, K+, Mg2+, Fe2+, Ba2+ slightly increased BbCSN-1 activity, while EDTA slightly inhibited BbCSN-1 activity.
Fig. 8. Deglycosylation and disulfide bonds analysis of BbCSN-1. (A) SDSPAGE analysis of BbCSN-1 treated with deglycosylation enzyme. Lane M, molecular weight marker. Fetuin is a control glycosylated protein provided in the protein glycosylation kit. (B) SDS-PAGE analysis of BbCSN-1 under reducing (with β-mercaptoethanol) and non-reducing (without β-mercaptoethanol) conditions. Lane M, molecular weight marker. Lane 1, with 12% β-mercaptoethanol (v/v) in loading buffer. Lane 2, without β-mercaptoethanol (v/v) in loading buffer.
revealed that BbCSN-1 could not hydrolyze (GlcN)2, (GlcN)3, or (GlcN) 4 (Fig. 7). However, chitosan oligosaccharides (GlcN)5 and (GlcN)6 were completely degraded by BbCSN-1. (GlcN)5 was hydrolyzed into (GlcN)2 plus (GlcN)3, and (GlcN)6 was hydrolyzed into mainly (GlcN) 3. These results indicated that for the degrading activity of BbCSN-1, the chitosan substrate should have a chain length over 4. Based on the degrading pattern of (GlcN)5 and (GlcN)6, BbCSN-1 may mainly release (GlcN)3 from the substrate by an endo-type cleavage. 3.7. Deglycosylation and disulphide bonds analysis of BbCSN-1 We performed enzymatic deglycosylation analysis of BbCSN-1 with the protein deglycosylation kit. After treated with deglycosylation enzyme, faster migration rate of a glycosylated protein Fetuin provided in the kit was observed in SDS-PAGE gel compared with the untreated protein. However, there is no significant change on the migration rate between non-treated BbCSN-1 and treated BbCSN-1, indicating no glycosylation happed in BbCSN-1 produced in P. pastoris. (Fig. 8A). To analysis whether disulfide bond(s) was (were) formed in BbCSN-1, this protein was subjected to SDS-PAGE analysis under reducing (with βmercaptoethanol in loading buffer) or non-reducing conditions (without β-mercaptoethanol). The results indicated BbCSN-1 treated with β-mercaptoethanol runs faster than that untreated protein
Fig. 7. Thin-layer chromatograph (TLC) of the reaction products of BbCSN1 from P. pastoris on chitosan oligosaccharides. The reactions were conducted at 30 °C for 20 min with glucosamine (G2), chitotriose (G3), chitotetraose (G4), chitopentaose (G5), and chitohexaose (G6) as the substrates. Lane M, standard mixture of chitosan oligosaccharides, ranging from glucosamine (G2) to chitohexaose (G6). 6
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(Fig. 8B). Therefore, disulfide bond(s) possibly exist in BbCSN-1. [5]
4. Conclusion
[6]
In the present paper, the active chitosanase BbCSN-1 of B. bassiana was successfully expressed in the P. pastoris expression system. Recombinant BbCSN-1 was purified by desalting size-exclusion chromatography. BbCSN-1 showed optimum activity at pH 5.0 and 30 °C and was stable at pH 2–8 and below 40 °C. Mn2+ significantly enhanced the activity of BbCSN-1, but Co2+ and Cu2+ showed inhibitory effects. The Km and Vmax values were 0.8 mg/ml and 3.9 g/min at 30 °C (pH 6.0), respectively. This study indicates that P. pastoris is an efficient heterologous expression system for the production of BbCSN-1, which can provide sufficient samples for further study of the enzymatic properties and even application.
[7] [8] [9] [10]
[11]
Acknowledgment Research was supported by grants from the Fundamental Research Funds for the Central Universities (XDJK2018C066 and XDJK2018AA006), the National Natural Sciences Foundation of China (31570137), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2019jcyj-msxmX0388 and cstc2018jcyjAX0073) and the program for innovation research team of Chongqing University (CXTDX201601012).
[12]
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Expression of a Beauveria bassiana chitosanase (BbCSN-1) in Pichia pastoris and enzymatic analysis of the recombinant protein
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Yu Liua,1, Yanling Lib,1, Sheng Tongb, Min Yuanb, Xiaoyun Wangb, Junyao Wangb, Yanhua Fanb,∗ a b
College of Biotechnology, Southwest University, 400716, Beibei, Chongqing, PR China Biotechnology Research Center, Southwest University, 400716, Beibei, Chongqing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Beauveria bassiana Chitosanase Pichia pastoris Purification Enzymatic characteristics
Chitosanase (EC 3.2.1.132) is an important chitosan-degrading enzyme involved in industrial applications. In this study, a chitosanase gene (BbCSN-1) from Beauveria bassiana, an insect fungal pathogen, was cloned and expressed in Pichia pastoris. The amount of BbCSN-1 in the fermentation broth of P. pastoris gradually increased after induction with methanol from one to 6 d, reaching 398 μg/ml on the 6th day. The molecular characteristics of BbCSN-1 were measured with colloidal chitosan as a substrate. The purified BbCSN-1 exhibited optimum activity at pH 5 and 30 °C and was stable at pH 2–8 and below 40 °C. The Km value of BbCSN-1 was approximately 0.8 mg/ml at 30 °C (pH 6.0). The activity of BbCSN-1 was significantly enhanced by Mn2+ but inhibited by Co2+ and Cu2+. These results indicated that BbCSN-1 from B. bassiana could be easily expressed in P. pastoris, which provided a basis for further study on its application.
1. Introduction Chitosanase (EC 3.2.1.132) can specifically catalyze the breakdown of the β-1,4-glucoside bonds in chitosan and release chitooligosaccharides (COSs) or glucosamine [1]. The first studied chitosanase was of microbial origin and can degrade Zygomycete cell walls [2]. Over the past 40 years, chitosanases have been found in different microorganisms and have shown potential for COS preparation and antifungal applications with their ability to degrade fungal cell walls [3–6]. The degrading products of chitosan by chitosanase, COSs, are alkaline oligosaccharides with multiple biological activities, such as anticancer, antioxidant, and immunostimulant effects. In addition, COSs also exhibit antifungal activity, which can antagonize plant pathogens and induce plant defense reactions [7,8]. Therefore, COSs have potential applications in medicine, agriculture, food, cosmetics and other fields and have attracted increasing attention. However, presently, only a limited number of chitosanases have been characterized, and enzymes from various sources have exhibited different enzymatic properties. To explore the application of chitosanase, it is necessary to characterize various chitosanases from different sources. Beauveria bassiana, an entomopathogenic fungus, has been widely used for the biocontrol of agricultural, forest, and urban pests [9]. In addition to inhabiting infected insect hosts, B. bassiana has other lifestyles, including saprophytes and endophytes [10]. Some hydrolases
have been found in this fungus, such as chitinases, proteases, and lipases [11]. These hydrolases have been verified to play vital roles in the fungal infection process against insect hosts by degrading host cuticles or being involved in antagonistic processes against phytopathogenic fungi [12,13]. Similar to chitinase and protease, B. bassiana chitosanase may also be involved in fungal pathogenesis or have antagonistic activity against other fungi. However, no chitosanase from entomopathogenic fungi has been characterized. In the genome of B. bassiana, there is a chitosanase-encoding gene (named BbCSN-1) that encodes a protein of 300 amino acids. To characterize BbCSN-1, the protein was heterologously expressed in the methylotrophic yeast Pichia pastoris, which can modify proteins by glycosylation, spatial folding, protease processing and the formation of disulfide bonds [14]. The expressed BbCSN-1 was purified from the culture supernatant, and the enzymatic properties were characterized with colloidal chitosan. 2. Materials and methods 2.1. Chemicals T4 DNA ligase was purchased from Promega (USA). All restriction endonucleases were from MBI (USA). The prestained protein marker was from Thermo (USA). The PCR primer was prepared by YingJun Biotechnology Company (Shanghai, China). Yeast extract and peptone
Corresponding author. E-mail address: [email protected] (Y. Fan). 1 Yu liu and Yanling Li contributed equally to this work. ∗
https://doi.org/10.1016/j.pep.2019.105519 Received 24 May 2019; Received in revised form 16 October 2019; Accepted 16 October 2019 Available online 18 October 2019 1046-5928/ © 2019 Published by Elsevier Inc.
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Fig. 1. Amino acid sequence and phylogenetic tree of BbCSN-1. (A) Amino acid sequence of BbCSN-1. (B) Phylogenetic tree of BbCSN-1 showing homologous proteins. (C) Construction map of recombinant expression vector pPIC9K-BbCSN-1 in P. pastoris.
2
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were from Sigma (USA). The DNA agarose recycling kit was from BioFlux (USA). PDA (potato dextrose agar) and PDB (potato dextrose broth) media were from BD (USA). The plasmid extraction kit was from Omega (USA). D-(+)-Glucosamine was purchased from Solar Biotechnology Co., Ltd. (Beijing, China). All chemicals used were of analytical grade. The protein deglycosylation Mix Ⅱ (P6044) was from BioLabs. (New England).
Superdex™ 75 pg, GE, USA). The column was equilibrated with 20 mM sodium acetate buffer (pH 5.4), and the protein was eluted with the same buffer at a flow rate of 0.7 ml/min. The purified protein was dialyzed against 20 mM sodium phosphate buffer (pH 5.0). N-terminal sequence of the purified BbCSN-1 was determined by Protein Sequencer PPSQ-31A (Shimadzu Corporation, Japan). Protein purity was analyzed based on densitometry (Bandscan soft).
2.2. Plasmids and strains
2.7. Enzymatic activity analysis of BbCSN-1
The A-T clone vectors pGEM-T and pEASY-Blunt were products from Promega and TransGen Biotech, respectively. The plasmid pPIC9K and P. pastoris GS115 strain were purchased from Invitrogen (USA). DH5α (E. coli) was purchased from TaKaRa (China).
To prepare colloidal chitosan, 1 g of chitosan powder was dissolved in 100 ml of 1% HCl. The pH of the chitosan solution was adjusted to 5.0 with NaOH. One milliliter of colloidal chitosan was collected by centrifugation (10,000 rpm, 5 min) in a 1.5 ml Eppendorf tube and washed with the same volume of Tris-HCl solution (1 M, pH 8.0) three times. Then, the substrate was resuspended in 1 ml of potassium phosphate buffer (20 mM, pH 5.0) and used for chitosanase analysis. The reaction mixture contained 100 μl of colloidal chitosan substrate, 100 μl of BbCSN-1, and 150 μl of potassium phosphate buffer (20 mM, pH 5.0). After reacting for 1 h at 30 °C, the reaction mixture was centrifuged, and 200 μl of supernatant was transferred into a new 1.5 ml Eppendorf tube, and 200 μl of DNS (3,5-dinitrosalicylic acid) and 100 μl of 1 M NaOH were added. After placing the tube at 95 °C for 10 min, the absorption value (OD540) was measured. D-Glucosamine was used to prepare a standard curve. One unit of chitosanase activity was defined as the amount of enzyme required to release 1 μM of D-glucosamine per hour. Three parallel groups were set up for each experimental group.
2.3. Expression plasmid construction of BbCSN-1 The intact coding region of BbCSN-1 in B. bassiana was obtained by PCR with primers Bbcsn-ye1 and Bbcsn-ye2 (Bbcsn-ye1: 5′-gaattccgcgacgtaccgtccaacgt-3′, Bbcsn-ye2: 5′-gcggccgcttaaccacgggcacacttgc-3’; the underline sequences are EcoRI and NotI sites, respectively) using B. bassiana genomic DNA as a template. There is no intron in this gene. The PCR product was cloned into the pGEM-T vector for DNA sequencing. The BbCSN-1 gene was excised from the pGEM-T vector with EcoRI, and the NotI site was used to insert the BbCSN-1 gene into the corresponding sites of the pPIC9K vector to produce pPIC9K-BbCSN-1. The recombinant fragment was transformed into E. coli DH5α, and the correct vector was verified by sequencing.
2.8. Characteristics of recombinant BbCSN-1
2.4. Transformation and selection of positive transformants
To determine the optimal reaction temperature, chitosanase activity was measured in potassium phosphate buffer (20 mM pH 5.0) at different temperatures (10, 20, 30, 40, 50 and 60 °C). To determine the optimal pH, the purified chitosanase activity was measured at different pH values (ranging from pH 2.0 to pH 10.0: pH 2.0–3.0, achieved with 0.05 M glycine-HCl buffer; pH 4.0–5.0, achieved with 0.2 M CH3COONa–CH3COOH buffer; pH 6.0–8.0, achieved with 0.1 M potassium phosphate buffer; and pH 9.0–10.0, achieved with 0.05 M glycine-NaOH buffer) for 30 min at 30 °C. The temperature and pH stability of BbCSN-1 were analyzed by using colloidal chitosan as a substrate. BbCSN-1 was pretreated for 30 min under different temperatures (10, 20, 30, 40, 50 and 60 °C) or different pH buffers as described above. Then, the chitosanase activity was measured with colloidal chitosan at pH 5.0 and 30 °C. To determine the optimal pH, the purified Bbchit1 activity was measured at different pHs (ranged from pH 3.0 to pH 10.0: pH 3.0-pH 6.0 was 0.1 M citrate–trisodium citrate buffer; pH 7.0–8.0 was 0.2 M potassium phosphate buffer; pH 9.0–10.0 was 0.05 M glycine–sodium hydroxide buffer) at 37 °C for 1 h. To determine the optimal reaction temperature, chitinase activity was measured in 0.2 M potassium phosphate buffer, pH 6.4, at different temperatures, 4, 15, 25, 35, 45, 55, and 65 °C, respectively.
pPIC9K-BbCSN-1 plasmid DNA was isolated and linearized with PmeI. The digested DNA was transformed into the yeast strain P. pastoris GS115 by electroporation according to the operating manual. In order to obtain yeast colonies with multiple copies of BbCSN-1, fast-growing positive transformants were selected in yeast peptone dextrose adenine (YPDA) medium supplemented with the antibiotic G418 (1.75 mg/ml) and confirmed by PCR. 2.5. Fermentation cultivation of BbCSN-1 in P. pastoris A colony of P. pastoris expressing BbCSN-1 was inoculated into 150 ml of buffered glycerol-complex medium (BMGY) (1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base with ammonium sulfate and without amino acids, 4 × 10−5% biotin, 100 mM potassium phosphate, pH 6.0, and 1.0% glycerol) grown at 28 °C in a shaker incubator. The cells were harvested by centrifugation and resuspended in 100 ml of buffered methanol-complex medium (BMMY) (1.0% yeast extract, 2.0% peptone, 1.34% yeast nitrogen base with ammonium sulfate and without amino acids, 4 × 10−5% biotin, 100 mM potassium phosphate, pH 6.0, and 1% methanol). The suspension was transferred to a sterile 1-L baffled flask and grown at 28 °C. To determine the time course of BbCSN-1 expression in P. pastoris, the fermented supernatant was collected daily (0–7 d) and analyzed for the expression of BbCSN-1 (SDS-PAGE), total protein concentration (Bradford method), activity of chitosanase and other experiments.
2.9. The influence of metal ions and EDTA on BbCSN-1 activity To evaluate the effects of metal ions on chitosanase activity, metal ions (5 mM), including MnCl2, FeCl3, CaCl2, BaCl2, CoCl2, MgCl2, CuSO4, FeSO4, KCl, NaCl, LiCl and EDTA were added to the reaction mixture. The relative activity was measured as the ratio between the chitosanase activity with metal ions and that without metal ions [15].
2.6. Purification recombinant BbCSN-1 from P. pastoris For purification of the recombinant BbCSN-1 from P. pastoris, the culture supernatant was concentrated by PEG 20,000 in a bag filter (MWCO 14000). The concentrated supernatant was directly subjected to desalting chromatography (AKTAprime plus, HiPrep™ 26/10 desalting chromatography column, GE, USA) equilibrated with 50 mM sodium acetate buffer (pH 5.4). The fractions containing BbCSN-1 were further purified by molecular sieve chromatography (HiLoad™ 16/600
2.10. The enzymatic kinetics of BbCSN-1 Colloidal chitosan (1–5 mg/ml) was used as a substrate to measure enzyme kinetics under standard conditions (20 mM potassium phosphate buffer, pH 5.0; 30 °C). Km and Vmax of BbCSN-1 were calculated 3
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Fig. 2. P. pastoris transformants of BbCSN-1 verified by PCR. Lane M, marker 2000; Lane 1, sterile water; Lane 2, pPIC9K plasmid; Lane 3, pPIC9KBbCSN-1 plasmid; Lane 4–6, different BbCSN-1 colonies. The arrow indicates the BbCSN-1 fragment.
by the Lineweaver-Burk double inverse method. 2.11. Analysis of chitosan oligosaccharides To understand the degradation pattern of BbCSN-1 against chitosan oligosaccharides, the TLC method was conducted as described by Choi [16]. 2.12. Enzymatic deglycosylation and disulphide bonds analysis of BbCSN-1
Fig. 4. Purified BbCSN-1 from P. pastoris culture supernatant. (A) Profile of BbCSN-1 purified by desalting chromatography. Peak 1 contained BbCSN-1 protein and chitosanase activity was detected (88.06U/mg). (B) Profile of BbCSN-1 purified by a molecular sieve. The fractions of peak 1–4 were collected and analyzed by SDS-PAGE. Activity of each peak was detected and only Peak 2 showed chitosanase activity (101.11U/mg).
BbCSN-1 was treated by enzymatic deglycosylation to determine whether this protein is glycosylated. Reaction protocols were followed by manufacturer's instructions. Control protein and BbCSN-1 were detected by SDS-PAGE respectively. To determine whether there are disulphide bonds in recombinant BbCSN-1, the recombinant protein was subjected for SDS-PAGE analysis under reducing (with 12% β-mercaptoethanol in loading buffer) and non-reducing conditions (without β-mercaptoethanol).
heterologously expressed in P. pastoris GS115. The expression vector pPIC9K-BbCSN-1 was constructed following the procedure described in the Materials and Methods (Fig. 1C). The obtained expression vector was transformed into P. pastoris cells by electroporation and grew on YPD plates containing 1.75 mg/ml G418 for screening multi-copy colony. Three rapidly growing positive transformants were selected and verified by PCR (Fig. 2).
3. Results and discussion 3.1. BbCSN-1 expression plasmid construction and selection of BbCSN-1 P. pastoris positive transformants The B. bassiana chitosanase gene BbCSN-1 encodes a protein of 300 amino acids (accession number: EJP64701) with an 18-aa N-terminal signal peptide predicted by Signal P. BbCSN-1 showed 99% identity to the homologous proteins from Cordyceps brongniartii (Fig. 1A and B). To analyze the enzymatic characteristics of BbCSN-1, BbCSN-1 was
3.2. Expression of BbCSN-1 in P. pastoris Three P. pastoris GS115 colonies containing BbCSN-1 were cultured in BMGY/BMMY culture medium and induced with 0.5% methanol to produce BbCSN-1. The supernatant of cultured P. pastoris cells was
Fig. 3. Time-course changes of BbCSN-1 expression in P. pastoris. (A) The expression of BbCSN-1 induced for different times was detected by 12% SDS-PAGE. Lane M, molecular weight marker; Lane P, fermentation broth of P. pastoris transformed with pPIC9K induced by methanol; Lane 1–7, fermentation broth of P. pastoris transformed with pPIC9K-BbCSN-1 induced by methanol for one day to seven days. Twenty microliters of pretreated sample was loaded in every lane. (B) The expression of BbCSN-1 on different induction days was measured by the Bradford method. 4
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detected daily by SDS-PAGE for the production of BbCSN-1. As the tested three colonies exhibited similar expression level of BbCSN-1 (data not shown), only one colony's results were shown here. Compared to the wild-type strain, a protein with a molecular mass of approximately 33 kDa, which was similar to the theoretical value of BbCSN-1 (Fig. 3A). The results showed that BbCSN-1 was expressed from the first day of induction with a protein amount of 60 μg/ml. The amount of BbCSN-1 gradually increased within 1–6 d and reached a peak at 398 μg/ml on the 6th day (Fig. 3B). These results indicated that BbCSN1 could be successfully expressed in P. pastoris GS115. The N-terminal sequence of the recombinant BbCSN-1 is EAEAYE, corresponding to the predicted cleave site of protease KEX2 signal cleavage. 3.3. Purification of recombinant BbCSN-1 from P. pastoris Fig. 5. SDS–PAGE analysis of BbCSN-1 after purification. Lane M, molecular mass standards indicated in kDa; Lane 1, blank vector sample; Lane 2, fraction after desalting chromatography; Lanes 3–6, peak from samples 1–4 after molecular sieve chromatography as shown in Fig. 4B.
To purify recombinant BbCSN-1 from P. pastoris, the supernatant was concentrated by 5-fold in a dialysis bag (MWCO 14 kDa). The sample was subjected to desalting chromatography (Fig. 4A). The fraction with chitosanase activity (Peak 1 in Fig. 4B) was further purified by molecular sieve chromatography (Fig. 4B, Fig. 5). After
Table 1 Purification of the BbCSN-1 from Beauveria bassiana expressed in P. pastoris. Purification steps
Total activity (U/L)
Total protein (mg/L)
Specific activity (U/mg)
Purification factor
Yield (%)
Culture supernatant Desalting Molecular sieve chromatography
7518.85 4100.71 2586.63
1371.74 46.57 25.58
5.48 88.06 101.11
1.00 16.07 18.45
100.00 54.54 34.40
Fig. 6. Effects of temperature and pH on chitosanase activity. (A) Effect of temperature. The chitosanase activity of BbCSN-1 was measured at temperatures ranging from 4 °C to 60 °C at pH 5.0 for 30 min. (B) Temperature stability of BbCSN-1. After BbCSN-1 was pretreated at temperatures ranging from 10 °C to 60 °C at pH 5.0 for 30 min, chitosanase activity was analyzed under standard conditions (pH 5.0, 30 °C). (C) Effect of pH. The chitosanase activity of BbCSN-1 was determined under a pH range from pH 2.0 to 10.0 at 30 °C for 30 min. The buffers used were 0.05 M glycine-HCl buffer (pH 2.0–3.0), 0.2 M sodium acetate-acetic acid buffer (pH 4.0–5.0), 0.1 M potassium phosphate buffer (pH 6.0–8.0) and 0.05 M glycine-NaOH buffer (pH 9.0–10.0). (D) pH stability of BbCSN-1. After BbCSN-1 was treated at temperatures ranging from pH 2.0–10.0 at 30 °C for 30 min, chitosanase activity was analyzed under standard conditions (pH 5.0, 30 °C). 5
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3.5. Enzyme kinetic parameters of purified BbCSN-1
Table 2 Effect of various metal ions and EDTA on chitosanase activity. Chemicals
Relative activity (%)
Chemicals
Relative activity (%)
None Mn2+ Fe3+ Na+ K+ Cu2+ Mg2+
100 233.3 ± 1.0 99.3 ± 1.2 118.0 ± 2.6 105.1 ± 5.5 51.9 ± 2.5 111.8 ± 4.4
Li+ Fe2+ Co2+ Ba2+ Ca2+ EDTA
102.9 ± 5.0 106.0 ± 2.8 50.9 ± 0.5 121.4 ± 1.5 99.2 ± 1.0 82.3 ± 4.0
To study the affinity between BbCSN-1 and the chitosan substrate, the Km of BbCSN-1 was detected. Based on the Lineweaver-Burk plotting method, the Km and Vmax of BbCSN-1 were approximately 1.52 mg/ml and 5.0 μg/min at 30 °C (pH 6.0), respectively. 3.6. Reaction pattern of purified BbCSN-1 The reaction products of purified BbCSN-1 were examined by TLC using several chitosan oligosaccharides as the substrates. The results
5 mM metal ions and EDTA were added to the enzymatic reaction mixture and chitosanase activity was measured with colloid chitosan as substrate (20 mM potassium phosphate buffer pH 5.0, 30 °C). The relative activity was the ratio of chitosanase activity with metal ions to the chitosanse without metal ion under the same reaction conditions.
purification, chitosanase in Peak 2 was obtained and protein purity is 80.4%. The specific activity of purified BbCSN-1 was determined to be 101.11 U/mg with an 18-fold increase compared to the original supernatant. The overall yield was estimated at 34.40%. (Table 1). 3.4. Enzymatic properties of purified BbCSN-1 The enzymatic properties of BbCSN-1 were measured with colloidal chitosan as a substrate [17,18]. The purified BbCSN-1 exhibited optimum activity at 30 °C and pH 5 (Fig. 6A, Fig. 6C). The stability analysis showed that BbCSN-1 was stable below 40 °C and that the relative activity was over 80% (Fig. 6B). However, the relative activity decreased to 60% after treatment at 50 °C or 60 °C for 30 min. In addition, BbCSN-1 was stable when the pH was 2–5; however, the relative activity gradually decreased with increasing pH (pH > 5) (Fig. 6D). The high pH conditions may cause the degradation of chitosanase or decrease chitosan substrate solubility, resulting in the decrease in enzyme activity. The influence of metal ions (5 mM) on the activity of BbCSN-1 was determined (Table 2). Mn2+ had a significant activation effect on the activity of BbCSN-1 and enhanced the activity of BbCSN-1 by 133%. However, Co2+ and Cu2+ had a strong inhibitory effect on the activity of BbCSN-1. With 5 mM Co2+ and Cu2+, the relative activity of BbCSN1 was decreased by 50%. Fe3+, Li+ and Ca2+ had no significant effect on the activity of BbCSN-1. Other metal ions, such as Na+, K+, Mg2+, Fe2+, Ba2+ slightly increased BbCSN-1 activity, while EDTA slightly inhibited BbCSN-1 activity.
Fig. 8. Deglycosylation and disulfide bonds analysis of BbCSN-1. (A) SDSPAGE analysis of BbCSN-1 treated with deglycosylation enzyme. Lane M, molecular weight marker. Fetuin is a control glycosylated protein provided in the protein glycosylation kit. (B) SDS-PAGE analysis of BbCSN-1 under reducing (with β-mercaptoethanol) and non-reducing (without β-mercaptoethanol) conditions. Lane M, molecular weight marker. Lane 1, with 12% β-mercaptoethanol (v/v) in loading buffer. Lane 2, without β-mercaptoethanol (v/v) in loading buffer.
revealed that BbCSN-1 could not hydrolyze (GlcN)2, (GlcN)3, or (GlcN) 4 (Fig. 7). However, chitosan oligosaccharides (GlcN)5 and (GlcN)6 were completely degraded by BbCSN-1. (GlcN)5 was hydrolyzed into (GlcN)2 plus (GlcN)3, and (GlcN)6 was hydrolyzed into mainly (GlcN) 3. These results indicated that for the degrading activity of BbCSN-1, the chitosan substrate should have a chain length over 4. Based on the degrading pattern of (GlcN)5 and (GlcN)6, BbCSN-1 may mainly release (GlcN)3 from the substrate by an endo-type cleavage. 3.7. Deglycosylation and disulphide bonds analysis of BbCSN-1 We performed enzymatic deglycosylation analysis of BbCSN-1 with the protein deglycosylation kit. After treated with deglycosylation enzyme, faster migration rate of a glycosylated protein Fetuin provided in the kit was observed in SDS-PAGE gel compared with the untreated protein. However, there is no significant change on the migration rate between non-treated BbCSN-1 and treated BbCSN-1, indicating no glycosylation happed in BbCSN-1 produced in P. pastoris. (Fig. 8A). To analysis whether disulfide bond(s) was (were) formed in BbCSN-1, this protein was subjected to SDS-PAGE analysis under reducing (with βmercaptoethanol in loading buffer) or non-reducing conditions (without β-mercaptoethanol). The results indicated BbCSN-1 treated with β-mercaptoethanol runs faster than that untreated protein
Fig. 7. Thin-layer chromatograph (TLC) of the reaction products of BbCSN1 from P. pastoris on chitosan oligosaccharides. The reactions were conducted at 30 °C for 20 min with glucosamine (G2), chitotriose (G3), chitotetraose (G4), chitopentaose (G5), and chitohexaose (G6) as the substrates. Lane M, standard mixture of chitosan oligosaccharides, ranging from glucosamine (G2) to chitohexaose (G6). 6
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(Fig. 8B). Therefore, disulfide bond(s) possibly exist in BbCSN-1. [5]
4. Conclusion
[6]
In the present paper, the active chitosanase BbCSN-1 of B. bassiana was successfully expressed in the P. pastoris expression system. Recombinant BbCSN-1 was purified by desalting size-exclusion chromatography. BbCSN-1 showed optimum activity at pH 5.0 and 30 °C and was stable at pH 2–8 and below 40 °C. Mn2+ significantly enhanced the activity of BbCSN-1, but Co2+ and Cu2+ showed inhibitory effects. The Km and Vmax values were 0.8 mg/ml and 3.9 g/min at 30 °C (pH 6.0), respectively. This study indicates that P. pastoris is an efficient heterologous expression system for the production of BbCSN-1, which can provide sufficient samples for further study of the enzymatic properties and even application.
[7] [8] [9] [10]
[11]
Acknowledgment Research was supported by grants from the Fundamental Research Funds for the Central Universities (XDJK2018C066 and XDJK2018AA006), the National Natural Sciences Foundation of China (31570137), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2019jcyj-msxmX0388 and cstc2018jcyjAX0073) and the program for innovation research team of Chongqing University (CXTDX201601012).
[12]
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