今天推送的文章发表在Bioresource Technology上的“Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-β-D-glucosamine from powdery chitin”，作者为中国农业大学的杨绍青教授。
Chitin, a linear polysaccharide composed of N-acetyl-D-glucosamine (GlcNAc) units linked by β-1,4 bonds, is one of the most abundant biopolymers in nature, especially in marine waste such as shrimp shells and crab shells, generating more than 1011 tons of solid waste per year. GlcNAc, as a monomeric unit of chitin, has been reported to have a variety of biological activities, such as anti-inflammatory, antitumor, and antioxidant activities. At present, GlcNAc is commercially produced by high-temperature and strong acid hydrolysis using chitin as raw material, but there are still shortcomings such as low yield, poor product stability, and environmental pollution risk. Enzymatic hydrolysis of natural chitin under mild conditions is a green and promising strategy for GlcNAc production, in which chitinolytic enzymes play a key role. The enzymatic biotransformation of chitin to GlcNAc requires the combined action of two types of enzymes, namely chitinase (EC 3.2.1.14) to produce N-acetylCOS by catalyzing the hydrolysis of β-1,4 bonds in chitin, while β-N-acetylglucosaminidase (GlcNAcase, EC 3.2.1.52) performs rate-limiting action to further hydrolyze N-acetyl-COS to GlcNAc. Increasing the specific activity of chitin by molecular modification is a candidate method to improve the efficiency of chitin hydrolysis. GlcNAcases can cleave N-acetylCOS and chitin with high crystallinity, thereby increasing substrate receptivity to chitinase. Aspergillus niger is an ideal cell factory because of its unique advantages such as high protein secretion efficiency, strong autolysis resistance, and safety, and may be more suitable for the construction of synergistic enzyme systems. In this study, a host strain, A. niger FBL-B (ΔglaA), capable of secreting high levels of endogenous GlcNAcase, was explored. At the same time, a dual-enzyme synergistic system was constructed. This system allows for the efficient production of designed chitinases from Bacillus pasteurii (PbChi70) as well as endogenous GlcNAcases for efficient transformation of powdered chitin.
Screening and expression of chitinase gene mutants
By error-prone PCR, a mutant library with a mutation frequency of 0.5% was constructed. A summary of the two rounds of screening and the number of mutants selected at each stage are shown in Figure 1. In the first stage of screening, a mutant library (containing 10,000 clones) is pre-screened using colloidal chitin plates. More than 45% of the clones exhibited clear spots, of which 215 were selected to exhibit clones comparable to or larger in size than the control (without mutations). Subsequently, selected clones were cultured and introduced into shake flasks (50 mL), and the secreted chitinases were characterized. Eventually, a mutant (mPbChi70) with significantly higher specific activity was obtained (Figure 1). DNA sequencing revealed three mutation sites (Asp50Val, Glu190Gly, and Phe272Trp) in mPbChi70. Multiple amino acid sequence alignment showed that Asp50Val and Phe272Trp were conserved in GH family 18 chitinases, while Glu190Gly was not. In order to analyze the effect of the three amino acids on the enzyme specific activity, three mutants were generated separately by site-directed mutagenesis. Mutations in Asp50Val, Glu190Gly, and Phe272Trp significantly increased the specific activity of the enzyme by 44%, 65%, and 34%, respectively.
mPbChi70的生化表征
The mutant (mPbChi70) was successfully expressed in its active form in Escherichia coli BL21. The recombinant enzyme is then purified to homogeneity and biochemically characterized. Compared to PbChi70, the optimal pH of mPbChi70 was increased from 5.5 to 6.5 (Figure 2a) and the optimal temperature was increased from 55 °C to 60 °C (Figure 2c), while no or minor differences were observed in pH and thermal stability (Figures 2b and d).
Compared to PbChi70, mPbChi70 significantly increases the specific activity of colloidal chitin and regenerated chitin by 2.44 and 1.45 times, respectively. However, no or minimal differences were observed on the other test substrates (Table 1). This mutation does not affect the Km value of the enzyme, but significantly increases the maximum velocity (Vmax) (1.7-fold) and catalytic efficiency (2.0-fold) (Table 2). The optimal conformations of mPbChi70 and PbChi70 and substrate were obtained by molecular docking. This mutation significantly increases the binding energy by a factor of 1.63 (Table 2).
In this study, PbChi70 was successfully engineered by directed evolution, and the specific activity of the selected mutant was increased by 2.4 times compared with that of the wild-type enzyme (73.21 U/mg) (Table 1). This value is significantly higher than that of other reported chitinases, which range from 2.1 to 66.2 U/mg, representing the highest specific activity of chitinases reported to date. Binding free energy calculations showed that the mutant (-10.04 kcal/mol) exhibited a higher binding affinity with GF5 compared to the wild-type (-6.15 kcal/mol). Interestingly, the mutation did not affect the Km value of mPbChi70, but significantly increased the Vmax value. The catalytic efficiency (kcat/km) is significantly increased by a factor of 2 compared to wild-type enzymes (Table 2).
Structural analysis of mPbChi70
As a member of GH family 18 chitinases, PbChi70 shares 79% highest amino acid sequence similarity with other structurally characteristic chitinases and has a classical (β/α) 8-TIM barrel fold. According to the model structure of PbChi70, two substitutions (Asp50Val and Glu190Gly) are located on the surface of the enzyme molecule (Figure 3a). The substituted Phe272 is located at the bottom of the catalytic trough at a minimum distance of 2.1 Å from the substrate. Figure 3b shows that when Phe is replaced by Try, the shortest distance to the docking small molecule increases to 3.4 Å, and the direction of the amino acid branching shifts from the inside of the catalytic tank to the outside. After directed evolution, the charge distribution in the region to the right of the catalytic trough shifted (Figure 3c).
Structural analysis revealed that there were three substitutions in the mutant mPbChi70 (Asp50Val, Glu190Gly, and Phe272Trp) (Figure 3a), and two substitutions (Asp50Val and Glu190Gly) located on the surface of mPbChi70 (Figure 3c). Two mutants were created separately by site-directed mutagenesis. Mutations in Asp50Val and Glu190Gly resulted in an increase in specific activity of 44% (43.3 U/mg) and 65% (49.7 U/mg), respectively. According to the protein surface charge analysis (Figure 3c), the negatively charged residues Glu190 and Asp50 at the catalytic trough boundary were replaced by the non-polar residues Gly and Val, respectively. These substituents may affect the local charge balance on the surface of the enzyme, thereby increasing the catalytic efficiency of the enzyme. The Phe272Trp mutation also resulted in a significant increase in specific activity by 34% (40.3 U/mg). To further analyze the role of this mutation site, a simulation analysis was performed using molecular docking (Figure 3B). Phe272 is located at the bottom of the catalytic trough and has a minimum distance of 2.1 Å from the docking small molecule substrate, while the minimum distance of the substituted Try272 increases to 3.4 Å. In addition, the branched chain of Phe272 is inside the catalytic tank, while the branched chain of Try272 is outside the catalytic tank. Therefore, it is speculated that the substitution can increase the specific activity of mPbChi70 by expanding the catalytic trough channel, reducing steric hindrance, promoting the substrate entering the central binding pocket, and promoting the release rate of the product.
Secreted expression of mPbChi70 in Aspergillus niger FBL-B (ΔglaA).
The mPbChi70 gene was successfully expressed in the parental strain A. niger FBL-B (ΔglaA), which was able to secrete endogenous GlcNAcase. Approximately 200 resistant transformants are obtained per μg of DNA. Select homologous transformants that produce high chitinase activity up to 8.03 U/mL and GlcNAcase activity up to 310 U/mL. The transformants were then fermented at high cell density, and the highest extracellular chitinase activity was 61.33 U/mL and protein concentration was 10.47 mg/mL after 144 h of incubation (Figure 4a). At the same time, GlcNAcase activity increased gradually, reaching a maximum yield of 353.1 U/mL at the end of fermentation (Figure 4a). In the initial stage (0~72 h), the growth rate of biomass increased rapidly, and the growth rate slowed down after batch feeding. Total sugar consumption increased steadily throughout the fermentation process, reaching 157.3 g/L at the end of fermentation (Fig. 4a). Check whether the chitinase and GlcNAcase secreted in the supernatant are correct by SDS-PAGE and zymogram analysis (Figure 4b).
Aspergillus niger is an attractive protein expression host that can secrete high levels of heterologous proteins. In this study, the engineered chitinase was transformed into the host strain A. niger FBL-B (ΔglaA), which is capable of secreting high levels of endogenous GlcNAcase, and a dual-enzymatic hydrolysis system was successfully constructed, which is the first specific dual-enzyme synergistic system for GlcNAc production in A. niger. The highest chitinase yield of 61.33 U/mL and GlcNAcase activity of 353.1 U/mL were obtained by high cell density fermentation in a 5 L fermenter (Figure 4a). The chitinase activity was significantly higher than that of other chitinases, second only to the chitinase of Bacillus licheniformis (168 U/mL) in Pichia pasteuris. The efficient hydrolysis of colloidal chitin in the initial phase of the two-enzyme mixture mainly produces (GlcNAc)2, and the (GlcNAc)2 formed at the end of the reaction is further converted to GlcNAc.
Conversion of GlcNAc from powdered chitin
The ability of enzyme mixtures to produce GlcNAc from chitin was evaluated. Scanning electron microscopy (SEM) was used to observe the morphological changes of powdered chitin during enzymatic degradation. Powdered chitin is in the form of large granules, and the structure is rough and dense. Treatment with mPbChi70 alone did not result in significant changes in the chitin structure of powder. However, the treatment of the double enzyme mixture significantly disrupted the powdered chitin granules and observed a large number of fragments. In addition, the dual-enzyme mixture treatment significantly reduced the crystallinity index (CrI) from 70.9% to 56.1%.
GlcNAc biotransformation of powdered chitin was carried out by mPbChi70 and a double enzyme mixture, respectively. When powdered chitin was hydrolyzed with mPbChi70 alone, the conversion rate was 18.34% (the products were mainly GlcNAc and (GlcNAc)2) (Figure 5a). The application of the two-enzyme mixture significantly improved the conversion rate, with a conversion rate of up to 71.9% (w/w) at 24 h and a GlcNAc content of 7.19 mg/mL (Figure 5b). GlcNAc is the only major product in the hydrolysate with a purity of up to 95% (Figure 5c). In this study, a dual-enzyme system composed of mPbChi70 and GlcNAcase co-expressed by Aspergillus niger was used to directly hydrolyze the powdered chitin to produce GlcNAc, with a conversion rate of up to 71.9% (w/w) (Fig. 5b). This method has the advantages of high conversion rate, easy sample operation, low cost, no need for special large-scale equipment, and strong practicability. The GlcNAc purity obtained in this study is as high as 95% (w/w) without purification (Figure 5c) and even close to the commercial GlcNAc (analytical grade, purity ≥ 96%) of Sigma Co.Ltd. Therefore, the strategies in this study may have great potential for application in the commercial production of GlcNAc.
DOI:10.1016/j.biortech.2023.130024
Link to the article: https://doi.org/10.1016/j.biortech.2023.130024