Investigation on the Complex Mutagenesis Selection of High-yield Nitrate Reductase Strain Staphylococcus simulans ZSJ6 and Its Enzymatic Properties

doi:10.13560/j.cnki.biotech.bull.1985.2022-1270

Abstract

Abstract:

Nitrate reductase can reduce the nitrate in natural meat products to nitrite, avoiding a sudden increase in its content due to direct addition of nitrite and reducing the chance of producing nitrosamines, which is very important for the quality and safety of natural meat products. Staphylococcus simulans D14 screened from sausage as the starting strain, and the different effects of single mutagenesis condition including UV, microwave, and lithium chloride mutagenesis, and two compound mutagenesis methods of UV and microwave combined with lithium chloride on the strains were investigated to obtain high-yield strains of nitrate reductase, and their enzymatic properties were initially explored by shake flask fermentation in order to provide strain selection for fermenters of natural meat products. The optimal conditions for UV, microwave and lithium chloride mutagenesis were determined: UV irradiation for 60 s, microwave irradiation for 80 s and lithium chloride concentration of 1.5%. The mutagenic strain S. simulans ZSJ6 was screened, and the enzyme activity of the strain reached 603.29 U/mg protein, which was 3.59 times higher that of the starting strain, and there was no significant change in the enzyme activity of the mutagenic strain after 8 consecutive passages（P>0.05）, indicating that its genetic stability was good. From the results of enzymatic properties, its optimal action temperature was 30℃, the optimal action pH was 7.5, and its stability was excellent, and the enzyme activity retained more than 90% after incubation at pH 7.5 for 2 h. Mg²⁺, Ca²⁺, and K⁺ all promoted enzyme activity, of which Ca²⁺ had the largest role in promoting the enzyme activity, which was 1.63 times higher than that blank group. Cu²⁺, Fe²⁺, Hg²⁺ and Mn²⁺ suppressed enzyme activity, among which Cu²⁺and Hg²⁺ had the maximum inhibitory action on the enzyme activity, and the enzyme activity was all inhibited to less than 30%. The above results provide a solution for the transformation of nitrate in natural meat products and the problem of high nitrite concentration brought by the use of nitrite at the initial stage of production, which has research value and potential for application.

Key words: Staphylococcus simulans, nitrate reductase, natural meat products, compound mutagenesis, enzymatic properties, nitrite, nitrate

ZHAO Sai-sai, ZHANG Xiao-dan, JIA Xiao-yan, TAO Da-wei, LIU Ke-yu, NING Xi-bin. Investigation on the Complex Mutagenesis Selection of High-yield Nitrate Reductase Strain Staphylococcus simulans ZSJ6 and Its Enzymatic Properties[J]. Biotechnology Bulletin, 2023, 39(4): 103-113.

Figures/Tables 14

Fig. 1 Growth and enzyme production curve of S. simulans D14

Fig. 2 Effects of UV irradiation on the lethality rate and positive mutation rate of S. simulans D14

Table 1 Screening results of UV mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
UV1-3	201.33
UV1-5	191.73
UV1-6	175.64
UV1-15	366.42
UV1-21	275.12
UV2-7	288.01
UV2-11	264.37
UV2-14	305.47
UV2-15	318.25
UV2-18	187.72

Fig. 3 Lethality rate and positive mutation rate of microwave mutagenesis

Table 2 Screening results of microwave mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
MW1-1	370.39
MW1-9	394.12
MW1-13	401.93
MW1-14	383.56
MW1-17	451.06
MW2-3	377.64
MW2-7	385.17
MW2-8	396.72
MW2-11	421.38
MW2-18	435.76

Fig. 4 Lethality rate and positive mutation rate induced by lithium chloride

Table 3 Screening results of mutagenic strains induced by lithium chloride

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
LiCl2	483.52
LiCl4	505.21
LiCl5	476.32
LiCl7	490.18
LiCl11	511.97
LiCl12	489.15
LiCl16	521.75
LiCl17	518.72
LiCl20	463.74
LiCl21	470.19

Table 4 Screening results of compound mutagenic strains

菌株编号Strain No.	酶活力Enzyme activity/（U·mg^-1 protein）
ZSJ1	473.36
ZSJ4	485.12
ZSJ6	603.29
ZSJ17	501.12
ZSJ18	556.71
ZSJ19	578.83
ZSJ23	511.97
ZSJ26	529.31
ZSJ30	536.75
ZSJ35	562.33

Table 5 The activity of nitrate reductase produced by mutant ZSJ6 in each generation

传代数 Passage number	酶活力 Enzyme activity/（U·mg^-1protein）	显著性 Significant level
1	603.29±0.01	P>0.05
2	604.88±0.02
3	602.19±0.03
4	604.18±0.02
5	599.56±0.02
6	600.12±0.01
7	603.73±0.02
8	601.17±0.01

Fig. 5 Effects of temperature on the activity of nitrate reductase produced by S. simulans ZSJ6

Fig. 6 Effects of temperature on the stability of nitrate reductase produced by S. simulans ZSJ6

Fig. 7 Effects of pH on the activity of nitrate reductase produced by S. simulans ZSJ6

Fig. 8 Effects of pH on the stability of nitrate reductase produced by S. simulans ZSJ6

Fig. 9 Effects of metal ions on the nitrate reductase activity * indicates a significant difference from the control group (P<0.05), no significant difference if not marked

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