Today's article published on carbohydrate polymers is "engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides", The corresponding author is Professor Yu Xiaobin of the School of Biological Engineering of Jiangnan University.
Chitosan oligosaccharides (chos) are biocompatible cationic polysaccharides with antibacterial activity because they adhere to the bacterial cell wall and inhibit the metabolism of bacteria. chos is a hydrolysate of chitosan, with low viscosity, high solubility, good moisture absorption, strong adsorption capacity, good biocompatibility and other characteristics, in cosmetics, pharmaceutical, agriculture and food industry has a wide range of applications. At present, the production methods of chos are chemical, physical and enzymatic. Among them, the enzyme-catalyzed degradation of chitosan or chitin has the advantages of mild reaction conditions, environmental sustainability, selectivity or high specificity, and has broad application prospects.
A variety of enzyme immobilization techniques (embedding, crosslinking and vector binding) have been developed to enable efficient production and separation of products, substrates and enzymes. In most cases, however, the enzyme needs to be purified prior to immobilization, and the product also needs to be tediously separated downstream from the substrate and residual enzymes. In addition, the reusability and stability of enzymes remains the main bottleneck in the application of enzyme immobilization in actual production.
Recently, strategies for fusing enzymes with carbohydrate binding modules (CBMs) have proven feasible in one-step purification and immobilization. CBM is a small domain widely present in carbohydrate enzymes that specifically recognize and bind carbohydrates, especially insoluble polysaccharides. Carriers used for immobilization are generally natural or food-safe polysaccharides such as cellulose, chitin, and microcrystalline cellulose. This strategy has been found to improve the properties of fusion enzymes, including their stability, activity, selectivity, and specificity.
In previous studies, the expression of cbm fusion enzymes was mainly carried out in E. coli, because E. coli always produces intracellular enzymes, so cells need to be destroyed. As an efficient expression system, Pichia yeast secretes only a small amount of its own protein, so it can greatly reduce the purification cost of enzymes in industrial applications. However, glycosylation in Pichia yeast in some cases reduces the substrate affinity of recombinant CBM. In addition, most of the immobilized carriers of CBM are cellulose, whose structure is also affected after binding to CBM. Therefore, finding a cbm with good compatibility with yeast expression systems is the main challenge to achieve one-step purification and immobilization of chitosanase.
BHCBM56 is a CBM of bacillus haloduransβ-(1,3)-glucanase and is a promising enzyme fusion option in immobilization. Agrobacterium water-insoluble linear β-(1,3)-dextran is a safe and inexpensive bhcbm56 immobilized carrier. In addition, we also found that chitosanase csn75 produced by Aspergillus fumigatus cj22-326 can effectively prepare chos with a higher degree of polymerization. Therefore, it is possible to utilize bhcbm56 as cbm, fuse csn75 at the c-end, express recombinant enzymes in Pichia yeast, and finally achieve one-step purification or immobilization of csn75.
In this study, we fused chitosanase into a carbohydrate binding module with bhcbm56 as cbm and csn75 as model enzyme to continuously produce the required chos. On the basis of efficient extracellular expression and high-density fermentation of Pichia, a chitosanase immobilized thermogel polysaccharide filler bed reactor (cicpr) was established, and the performance test of continuous production of chos was carried out. Different reaction conditions (e.g., chitosanase amount, flow rate, substrate concentration) were optimized. During this process, the stability and reusability of CICPR were also evaluated.
Expression and characteristics of csn75, csn75-cbm and csn75-2cbm
Recombinant enzymes csn75-cbm and csn75-2cbm at p. Successfully expressed in pastoris gs115. SDS-page analysis confirmed the extracellular expression of csn75, csn75-cbm, and csn75-2cbm enzymes (fig. 1a). The molecular weights of the three enzymes were predicted to be 25.5 kda, 35.2 kda and 47.0 kda, respectively. In addition, there was no significant difference in protein content in the three fermentation supernatants, indicating that the extracellular expression of the three enzymes was similar (fig. 1b). But the activity of the recombinant enzymes csn75-cbm and csn75-2cbm decreased to 75.7% and 25.4% of the native csn75, respectively. Therefore, in order to maintain high enzyme viability and achieve immobilization of the enzyme, the authors chose csn75-cbm for further study.
Active center analysis of CSN75 and CSN75-CBM
The three-dimensional structure of the native csn75 (fig. 2a) and recombinant enzyme csn75-cbm (fig. 2b) was predicted based on amino acid sequences. The possible catalytic sites for chitosan hydrolysis (d143 and e152) are represented in pink, and the possible curdlan binding sites in cbm (y266, d268 and h270) are highlighted in red (fig. 2b). The recombinant enzyme csn75-cbm has both the catalytic activity of chitosan hydrolysis (provided by csn75) and the binding capacity with thermogel polysaccharides (provided by cbm). These two functions provide the possibility for further purification and immobilization of the recombinant enzyme csn75-cbm.
High-density fermentation and enzymatic profiling of CSN75 and CSN75-CBM
SDS-page analysis of high-density fermentation broth supernatants by recombinant plasmids gs115-ppic9k-csn75 (fig. 3a) and gs115-ppic9k-csn75-cbm (fig. 3b) showed that csn75 and csn75-cbm enzymes were the main proteins in their supernatants. Enzymatic profiling is used to evaluate the affinity of the recombinant enzyme csn75-cbm for chitosan. The bands of crude and purified csn75 and csn75-cbm on the denatured sds-page gel (fig. 3c) are consistent with those on the enzyme spectrum gel (fig.3d), indicating that the introduction of cbm into csn75 does not affect the affinity of the recombinant enzyme to the substrate. Only crude and purified csn75 and csn75-cbm show clear spots (fig.3d) on enzymatic gels, suggesting that csn75 and csn75-cbm are the only proteins with chitosanase activity in their fermentation supernatants.
Comparison of enzymatic properties of CSN75 and CSN75-CBM
To evaluate the effect of fusion CBM on the properties of the csn75 enzyme, their catalytic activity, ph and thermal stability were compared. In acetic acid buffer (0.2 m, ph6.0), the activity of csn75-cbm at 50 °C was 10.26±0.77 u/ml, slightly lower than the activity of csn75 (13.56±0.86 u/ml). The optimal phph of csn75-cbm is 5.0, while the optimal phph of csn75 is 5.5 (fig. 4a). Both csn75 and csn75-cbm exhibited good catalytic stability in the ph 4.5-8.0 range, and the relative activity was above 90% (fig. 4b). The optimal temperature for csn75-cbm is 50-60 °C, slightly lower than that of csn75 (55-65 °C) (fig. 4c), indicating that the temperature of csn75-cbm is milder than that of csn75. After 2 h incubation at 50 °C, the relative activity of csn75-cbm was still 82.60%, while the relative activity of csn75 was only 70.11% (fig. 4d).
Adsorption properties of thermogel polysaccharides to csn75-cbm
Fe-sem analysis showed that the swollen gel particles (about 300 μm) were much larger than the undisplexed gel particles (nearly 10 μm) (fig. 5a, b). The swollen gel particles have good fluidity and looseness and are a good carrier. To further confirm whether the thermogel polysaccharides can adsorb the recombinant enzyme csn75-cbm, fitc-stained hot gel polysaccharides were analyzed with clsm. Before the recombinant enzyme csn75-cbm (fig. 5c), there was almost no green fluorescence on the surface of the gel particles, but after the recombinant enzyme treatment (fig. 5d), a strong green fluorescence was observed on the surface of the gel particles. This indicates that the recombinant enzyme csn75-cbm was successfully adsorbed by thermogel polysaccharides.
For better immobilization, the maximum adsorption capacity is evaluated by solid-state depletion. After the recombinant enzyme csn75-cbm is immobilized, the hot gel polysaccharide volume increases by a factor of 5 (fig. 6a) and the filler bed appears milky white (fig. 6b). Solid-state depletion experiments showed that the adsorption curve of csn75-cbm to thermogel polysaccharides conformed to the langmuir adsorption isothermal formula, and the correlation coefficient r2=0.9777(fig. 6c). The pichia pastoris extracellularly expressed csn75-cbm (fig. 6d) has the dual functions of "cutting" and "anchoring", contributed by "csn75" and "cbm", respectively. This allows csn75-cbm to be purified and immobilized in one step by thermogel polysaccharides (fig. 6e). CICPR was prepared by filling the csn75-cbm into a thermogel polysaccharide filler bed reactor. Chitosan is hydrolyzed by the recombinant enzyme csn75-cbm as it flows through cicpr (fig. 6f).
Performance of immobilized csn75-cbm in a filler bed reactor
In order to save the amount of enzymes used while meeting the production requirements, the load of enzymes was studied. The results showed that when the load of the enzyme was less than 40 U (fig. 7a), cicpr could not completely hydrolyze the substrate, while when the load was greater than 60 U, the DP of chos was closer. Therefore, the optional load of the enzyme should be 60-100 u, not exceeding the maximum load of cicpr (200 u).
The authors further investigated the effect of chitosan concentration on the degree of chos polymerization in the hydrolysate. A decrease in substrate concentration leads to a decrease in the polymerization of chos in the hydrolysate (fig. 7b). In continuous production processes, flow rates have a great impact on production efficiency. As the flow rate decreases, the degree of polymerization of chos in the hydrolysate decreases (fig. 7c). These results prove that CICPR is feasible for continuous production of ideal chos as long as the process parameters are adjusted. To obtain chos with ideal dps, qualitative and quantitative determinations were performed on three hydrolysates under given conditions (fig. 8a, b, and c). The results showed that under the conditions I., II., III., the main products in the hydrolysate were 2-5 (hydrolysis rate 97.75%), 3-6 (75.45%) and 3-7 (73.2%) chos (fig. 8d). After freeze-drying, the product is available in milky white, pale yellow and brownish yellow (fig. 8e) under three conditions. CICPR can achieve efficient production of chos with ideal dp. In addition, the immobilization of this enzyme also achieves a heterogeneous catalysis of chitosan hydrolysis in cicpr.
The stability and reusability of cicpr
The stability and reusability of cicpr are important indicators to evaluate its feasibility in continuous production, especially in large-scale industrial production. After 5, 10, 15 and 20 d at 4 °C, the catalytic activity retention rate of Cicpr was 97.8%, 95.6%, 92.0% and 88.3%, respectively. After 20 days, the catalytic activity of CICPR remained above 88% (fig. 9a). This shows that cicpr has good stability and is conducive to its flexibility in secondary application in actual production. To further verify the reusability of cicpr, the residual amount of reducing sugars (fig. 9b) was detected. The results show that CICPR still maintains more than 86% hydrolytic activity after 5 cycles, indicating that CICPR shows good stability and reusability in the actual production process.
In order to achieve continuous production, batch fermentation of feed is carried out, including three steps of batch feeding, balance and hydrolysis of enzymes. The concentration of chos in the hydrolysate was detected with hplc (fig. 9c) and qtof-ms (fig. 9c). QTOF-MS analysis showed that the hydrolysate of csn75-cbm is a highly polymerizable chitosan oligosaccharide, comprising (glcn)2 (m/z341.16), (glcn) 3 (m/z502.22), (glcn) 4 (m/z663.29), (glcn) 5 (m/z824.36) and (glcn) 6 (m/z985.43), and low polymerization chitosacose, including (glcn) 2-glcnac (m/z). Mixture of 544.24) and (glcn) 3-glcnac (m/z705.29). It was observed that cicpr could be renewed after batch fermentation of feeds, so that chos could be produced continuously.
In this paper, the authors expressed a cbm fusion chitosanase (csn75-cbm) using pichia pastoris outside of cells. CSN75-CBM exhibits higher thermal stability than csn75 and has the ability to bind specifically to thermogel polysaccharides, enabling one-step purification and immobilization. Based on this function, a fixed csn75-cbm filled bed reactor (CICPR) was built for the continuous production of chos with ideal DP. By adjusting the ratio of enzymes to chitosan or the flow rate of chitosan, chitosan with a degree of polymerization of 2 to 5 (hydrolysis rate of 97.75%), 3 to 6 (75.45%), and 3 to 7 (73.2%) was continuously produced. CICPR's good stability and reusability make it possible to apply it to actual production on a large scale and will greatly reduce the cost of downstream bioprocessing, and will also provide more inspiration for enzyme-catalyzed continuous biosynthesis of green chemicals or biodegradable environmental pollutants.
Finishing: Sun Xiao
Article information:
pmid：34561008
doi:10.1016/j.carbpol.2021.118609
Article link: https://doi.org/10.1016/j.carbpol.2021.118609