Characteristics and mechanism of a novel chitinase mutant from Trichoderma harzianum with enhanced activity


 Background: Chitinase from Trichoderma harzianum could inhibit various chitin-containing pathogens. In this study, a mutant of Chit42 (Chit42m) with high enzyme activity was established by error-prone PCR method.
Results: The Chit42m with three amino acids substitutions (D100G/I166V/A382P) was obtained from 1230 colonies. The activity of the Chit42m was enhanced by 1.26 times and affinity increased by 3.2 folds. The Chit42m showed the optimum temperature was 50 °C and pH 7.5.
Conclusion: Structural model and molecular docking analysis suggested that A382P may affect the catalytic activity of enzymes by affecting the conformation of key residues D169 and E171 related to catalysis, while I166V probably affects the affinity of chitin and enzymes by influencing the conformation of important substrate binding residues R52 and Y293. D100G had little effect on the changes of enzyme activity. This would put insight into the study of the site-specific mutations and provide promising gene material for the directed evolution chitinase.

to generate random mutations. Approximately, 1230 colonies of random mutagenesis libraries were submitted to initial screening. After the clones were incubated at 30 °C for 96 hours, the clear hydrolysis zones of different sizes formed on SC-U-beta plate was shown in Fig. 1. The standard for initial screening was the diameter ratio of transparent halo and colony. It was observed that a positive clone displayed the largest hydrolytic zone. The sequence variant was identified with four base substitutions (A299G, T300G, A496G and G1144C), compared with that of Chit42wt, resulting in the substitutions of three amino acids (D100G, I166V, A382P).

Comparison of the enzymatic properties of Chit42wt and Chit42m
The enzyme activity and Kinetic properties The activity of Chit42m was 0.48 U/g, 1.26 folds higher than that of Chit42wt with 0.38 U/g when the incubation time was 48 min, as was shown in Fig. 2. The kinetic analysis results revealed that Km of Chit42m was 8.47 mg/ml, Chit42wt was 27.4 mg/ml, indicating that affinity of the Chit42m was 3.2 folds higher than that of the Chit42wt.

The physicochemical properties
Temperature and pH were of significance to the activity and stability of chitinase. Fig. 3(a) revealed that there was no significant difference in temperature on chitinase activity. Interestingly, they demonstrated similar activity profiles at temperatures ranging from 40-50 °C and 60-70 °C, with optimal activity at 50 °C. For further characterization, the thermostability profile was also measured ( Fig. S1). The activity of Chit42m was always higher than that of the Chit42wt in the initial 50 min, but then decreased sharply, 80 % of the activity remained even after 70 min incubating at 50 °C, The relative activities of the Chit42wt and the Chit42m in the range of pH 3-8.5 were measured, exhibiting maximum activity at pH 7 and 7.5, respectively, as observed in Fig. 3(b). They both showed the uptrend of activities in acid conditions and downtrend in alkaline conditions, indicating that the enzyme was active within the neutral solution. The Chit42m demonstrated higher activity than the Chit42wt when the pH was 3-6.5 and 7-8.5, indicating that the stability of chit42m was enhanced significantly. The optimum pH of the Chit42m was 7.5.

Effect of metal ions on enzyme activity
In this study, Mg 2+ , K + slightly enhanced the activity of chitinase. Ca 2+ almost had no effect on the enzyme, Ba 2+ was the most effective to activate the enzyme, with the Chit42m increased by 76.3 %, higher than that of Chi42wt by 45.7 %.
Prediction of the mechanisms of the enhanced activity and affinity Chit42wt (EC 3.2.1.14) belongs to the glycoside hydrolase (GH) families 18, which contains a (β/α) 8 TIM barrel and an extra α/β domain. Homology models and docking results showed that Chit42wt contains a long substrate-binding groove similar as in other family 18 chitinases. Fig. 4 (a) showed the Chit42wt and substrate (chitohexaose) complex structure of molecular docking. Chitohexaose was bound into six sugar-binding subsites in Chit42wt, numbered from -4 (non-reducing end) to +2 (reducing end) with hydrolysis taking place on the glycosidic bond between the -1 and +1 subsites.
The catalytic residue Glu171 located in the -1 subsite. Trp131 and Trp378 stacked with the sugar rings in the -1 and +1 subsite, respectively. The mutated residues (D100, I166 and A382) of Chit42 did not directly interact with sugars. Molecular docking studies showed that the binding free energy of Chit42wt and Chit42m were -12.88 and -14.74 kcal/mol, respectively, which suggested that the variant showed higher affinity for the substrate. The superimposition of the structures of Chit42wt and the Chit42m after 1ns MD simulations was shown in Fig. 4 (b). The overall structure of the Chit42m was similar to that of the Chit42wt enzyme. Though residues at position 100, 166 and 382 did not directly contact with the substrate, the mutation was predicted to change the conformations of several residues surrounding the substrate involved in substrates binding and recognition (Fig .5). In the binding site, the side chains of W131, W133, F245, Y293 and W315 in the Chit42m mutant showed a significant shift relative to the Chit42wt thus affecting the binding of the substrate. In mutant, E171 was more closer to the bound GlcNAC unit at subsites -1 and +1, and two hydrogen bonds were formed between carboxyl oxygen of E171 and sugar units at -1 site and +1 site, respectively. While in Chit42wt, E171 only formed one hydrogen bond with +1 site sugar. The dynamic changes caused by structural changes were consistent with the experimental results.

Discussion
As for the mutantion site of chitinase, the substitution N35D of chitinase Pachi improved nematicidal activities by random mutagenesis [20]. In contrast, the three mutation sites produced by T. harzianum with higher activity and stability in this work had never been reported before. The number of point mutations observed was identical with the ideal mutagenesis of 1.5 to 5 base substitutions per 1000 bp [21], which could be expected from the PCR condition. Colloidal chitin with the highest activity to chitinase compared with other substrates was used as the substrate of chitinase, although chitosan had higher solubility [22].
The optimal temperature of Chit42m was 50 °C, which was the same with the chitinase from Bacillus licheniformis [23] and chitinase from Streptomyces spp. [24]. Moreover, 80 % of the activity remained even after 70 min incubating at 50 °C indicating that the thermostability was much better than the chitinase from Bacillus licheniformis with the activity decreasing by 50 % at 50 °C [25]. The optimum pH of Chit42m was 7.5, which was consistent with the chitinase from Bacillus sp. [26], similar with the chitinase from Stenotrophomonas maltophilia with the optimally active at pH 7 [27].
Metals ions performed different functions on enzyme in catalysis of redox reactions based on if they bind to the cofactors to form stable units [28]. Fig. S2 showed the effect of metal ions on enzyme activity. The chitinase could be stimulated by K + , Mn 2+ , Mg 2+ and Ca 2+ [29]. The effect of Ca 2+ is similar with thatit had no influence on the chitinase of Bacillus spp [30]. Ba 2+ also played active function to the chitinase from Streptomyces roseolus [ 6]. Presumably, the enhancement of Chi42wt and Chit42m activity arised from the conformation change of the enzyme and affected the binding of the enzyme and substrate, the mechanism of which would be studied henceforth. Co 2+ often conducted an inhibitory performance on the enzyme [24].
The mechanism of its activity enhancement was revealed by molecular docking. Comparisons of these three mutation sites and surrounding residues showed in Fig .6. D100 of Chit42wt sits in loop β6α4 (residues 96-100), far away from binding groove of Chit42wt. D100 could form stable hydrogen bonds with S97, N99, N104 and Y106 in Chit42wt Fig. 6 (a). When Asp was mutated to Gly, it broke the original hydrogen bonds, causing this loop region more instable than Chit42wt, but these residues had no concern with substrates binding, which suggested that D100G was not the cause of the change in enzyme activity. In contrast, Chitinase from Enterobacteriaceae could not do the enzyme-substrate model, the analysis of its activity and catalytic property could only depend on its binding domain from its 3D structure as lacking of its crystal structure. [31]. Like other family 18 chitinase, Chit42wt has a sequence motif DXXDXDXE (residues 164-171 of Chit42) at strand β5. I166 lies in this motif, and formed hydrophobic interactions with residues L126, I155 and I168 in Chit42wt. In mutant, though Val was also hydrophobic residue, its side-chain forcing side-chains of L126, I155 and I168 deflection Fig.   6 (b). Furthermore, I166V mutation also affected side-chains of D167, D169 and E171 in the conserved motif. E171 probably acted as the proton donor in the reaction and D169 was putative stabilizer of the positively charged oxazolonium ion intermediate [32,33]. Thus, I166V indirectly affected catalysis of Chit42. Residue A382 in Chit42wt was surrounded by residues R52, I292, Y293, D321, Y322, K323, E379, A380, S381, D383 and K384 etc., of which R52 and Y293 were important for interacting with the substrate Fig. 6 (c). When Ala was mutated to Pro, it was possible to make the region P382 sited more rigid, and affected conformation of R52 and Y293, therefore affected the binding of substrate. These results together provided important insights into structure-function relationship of Chit42m. Amino acid substitution at position 382 was determined to be critical for enzyme activity, while the residue at positions 199 and 116 had only marginal effect. Taken as a whole, the enhanced enzymatic activity of Chit42m was ascribed to the conformation changes of three key residues, which has reference significance for the further modification.

Conclusion
In summary, in order to obtain high enzymatic activity of chitinase, error-prone PCR was used to create Chit42 random mutations. The results identified that Chit42m mutant with three residues mutated showed high enzyme activities. Structural prediction showed that mutations could affect the conformation of the key residues that Chit42m had higher substrate affinity than the wild type. In conclusion, the Chit42m mutant has high enzymatic activity which may as a novel resource for related research and practical applications.

Characterization of Chit42wt and the mutant Chit42m
The optimal temperature of Chit42wt and the Chit42m was tested after incubating for 10 min within      Comparisons of mutation sites and surrounding residues. The structures of Chit42wt and Chit42m were showed as ribbon and selected residues were showed as stick. The corresponding hydrogen bonds were labeled as dashed lines in green.