[논문]Heterologous Expression of Recombinant Transglutaminase in Bacillus subtilis SCK6 with Optimized Signal Peptide and Codon, and Its Impact on Gelatin Properties

Wang, Shiting; Yang, Zhigang; Li, Zhenjiang; Tian, Yongqiang

doi:10.4014/jmb.2002.02049

Heterologous Expression of Recombinant Transglutaminase in Bacillus subtilis SCK6 with Optimized Signal Peptide and Codon, and Its Impact on Gelatin Properties 원문보기

Journal of microbiology and biotechnology, v.30 no.7, 2020년, pp.1082 - 1091

Wang, Shiting (Key Laboratory of Leather Chemistry and Engineering (Sichuan University), Ministry of Education) , Yang, Zhigang (Chengdu Jinkai Bioengineering Co., Ltd.) , Li, Zhenjiang (Chengdu Jinkai Bioengineering Co., Ltd.) , Tian, Yongqiang (Key Laboratory of Leather Chemistry and Engineering (Sichuan University), Ministry of Education)

Abstract ▼ AI-Helper

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

주제어

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문제 정의

75 U/mg without any chemical inducer. In conclusion, this study provides a new strategy for the efficient production of MTG that could be used in salt-containing foods.

제안 방법

The DNA sequence encoding MTG was amplified using forward primer (5'-CGCGGATCCTCGCCACCGGCAGTGGCAGTGGCAGCG-3') with a BamHI recognition sequence and reverse primer (5'-CTAGCTAGCTCACGGCCAGCCCTGTGTCA-3') with a NheI recognition sequence (GeneBank Accession No. MN700931).
To improve the extracellular expression of MTG, three signal peptides (SP_YncM, SP_NprE, and SP_Vpr) from the Sec pathway and four signal peptides (SP_YwbN, SP_LipA, SP_AmyX, and SP_WapA) from the Tat pathway were selected to construct recombinant MTG plasmid. The recombinant strains with different signal peptides were incubated in the fermentation liquid medium to determine extracellular MTG activity for 60 h.

대상 데이터

MTGase-producing bacterium Streptomyces sp. TYQ1024 (GeneBank Accession No. MN606211) was maintained in our laboratory. Escherichia coli DH5α (Vazyme, China) was used for vector construction.

이론/모형

The Michaelis-Menten equation was used to determine the kinetic parameters of MTG, which were examined under the substrate of CBZ-Gln-Gly from 5 to 30 mmol/l (pH 6.0). The values of Michaelis constant (K_m) and maximum velocity (V_max) of MTG were calculated from the Lineweaver-Burk plot.

성능/효과

In this study, codon and signal peptide optimization improved the extracellular expression of MTG in B. subtilis SCK6 (Ync M-TGSO) which showed the highest extracellular MTG activity (6.7 U/ml, 2.86-fold). The enzyme showed maximum activity at pH 8 and 50°C.
2 U/ml MTGase activity [31]. In this study, the MTGase expressed in B.subtilis SCK6 exhibited a secretion efficiency of 6.7 U/ml, showed a large yield advantage and effectively reduced the cost of industrial production.
The reductants DLDithiothreitol (DTT, 142.42 ± 1.84%, 5%) and β-Mercaptoethanol (β-ME, 160.18 ± 1.5, 5 mm) enhanced MTG activity, while PMSF and SDS inhibited MTG activity.

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