BACKGROUND

As mentioned in our design page, as the most critical enzyme in our multi-enzyme complex, manganese peroxidase plays a very important role. However, during our experiments, we found that the stability of wild-type MnP (wtMnP, BBa_K500001) was not good enough. Therefore, we tried to enhance its stability through directed evolution.

DESIGN

We made rational designs based on thermostability. By introducing parameters including salt bridge, secondary structure, RMSF, RMSD, protein gyration radius (GYRATE), hydrogen bond number, solvent accessibility surface area (SASA) and so forth, comprehensive analysis of these datas, we established a single point mutation database (File 1) based on FoldX, also we established a multifunctional enzyme library (File 2) based on Rosetta and Funclib for activity analysis. It is expected that manganese peroxidase with higher temperature stability can be obtained.

Table 1 Mutation sites of ten mutants with the smallest ΔΔG according to computational simulation.

Mutant No.	Position of amino acids	Mutation	ΔΔG(kcal/mol)
1#	74	E-P	-2.255
2#	74	E-M	-2.059
3#	182	D-I	-1.711
4#	182	D-V	-1.637
5#	182	D-T	-1.544
6#	232	S-P	-1.306
7#	74	E-L	-1.239
8#	78	S-P	-1.173
9#	183	Q-P	-1.079
10#	182	D-C	-0.9288

By analyzing the established single mutation library, ten mutants with the smallest ΔΔG were selected for stability verification (Table 1). We applied site-directed mutagenesis ( click here to see primer sequence) to construct our Manganese peroxidase mutants.

RESULT

Finally, mutants 1^#, 2^#, 5^#, 6^#, 7^#, and 8^# were successfully expressed in Pichia pastoris. The enzyme activity of the mutants and wtMnP was compared by monitoring the oxidation of 2,6-dimethoxyphenol (2,6-DMP) at 469 nm. After comparison, we found that mutant 2^# performed outstandingly.

Fig. 1 Thermostability of mutant 2^#. The initial MnP activity before incubation was set as 100%.

Fig. 2 Effect of temperature on the stability of mutant 2^# and wtMnP after 6 h incubation.The initial MnP activity before incubation was set as 100%. r.t. refers to room temperature. ^*P < 0.05, ^**P < 0.01.

Firstly, we detected the changes in the stability of mutant 2^# over time at different temperatures (Fig 1). After 2 h incubation, the enzyme activities of mutant 2^# incubated at different temperatures reduced to varying degrees. It is worth noting that in the subsequent incubation, mutant 2^# enzyme activity at 37℃ was improved. Compared with wtMnP, the stability of mutant 2^# at r.t., 50℃, and 60℃ has been significantly improved (Fig 2).

Fig. 3 Effect of pH on the stability of mutant 2^# and wtMnP after 12 h incubation. The initial MnP activity before incubation was set as 100%. ^**P < 0.01.

Considering that manganese peroxidase may be applied under various complex environments in reality, we subsequently tested its pH stability.

After incubation, the stability of mutant 2^# and wtMnP under different pH condition displayed similar tendencies (Fig 3). At pH = 4, the stability between the two enzyme showed a significant difference as mutant 2^# demonstrates an improved activity.

All in all, we screened single point mutations of manganese peroxidase. Mutant 2^# outcompetes other screened mutants, displayed a significant improvement regarding thermostability, and was basically consistent with wtMnP in other aspects. In conclusion, mutant 2^# is more suitable for use in higher temperature environments than wtMnP, while its applications under other physiochemical conditions will not be impaired.