Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from <i>Myxococcus</i> sp.V11

doi:10.13523/j.cb.1907049

China Biotechnology

2020, Vol. 40

Issue (3): 79-87 DOI: 10.13523/j.cb.1907049

Orginal Article

Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from Myxococcus sp.V11

ZHAO Xiao-yan¹,CHEN Yun-da¹,ZHANG Ya-qian¹,WU Xiao-yu^1,²,WANG Fei^1,^2,^**(

),CHEN Jin-yin²

1 College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang 330045, China
2 Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province, Nanchang 330045, China

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Abstract

The trehalose synthase (EC 2.4.1.245) from Myxococcus sp.V11 (TreS II) catalyzes the reversible interconversion of maltose and trehalose. The high catalytic activity and high conversion rate of maltose into trehalose of TreS II indicate that it has potential application in industrial production of trehalose. However, the thermal instability of TreS II limits its wide application in trehalose production. Objective:The effects of amino acid residues mutations on the thermal stability, optima of pH and temperature, and specific activity of TreS II were studied by site-directed mutagenesis. Methods: Site-directed mutation experiment of the two possible metal ion-binding sites (A283 and Y537) and the three sites (Q3, W374 and R449) in two regions which may correlate with thermostability by using overlapping PCR were performed. Mutants of A283R, Y537H, Q3D, W374D and R449Q were heterogeneous expressed in E.coli BL21(DE3). At the same time the specific activity, the optimum reaction temperature, the optimum pH and the thermal stability of mutants were compared with wild-type strain. Results: Mutation of Q3D, W374D, R449Q, A283R and Y537H enhanced the thermal stability, but did not affect the pH and temperature optima. Only the mutant R449Q reduced the specific activity. The modified enzymes A283R and Y537H showed 68% and the mutants Q3D, R449Q, W374D showed 35% of maximal activity after incubating in maltose substrate for 3h at 60℃ compared to only 20% activity for wild-type enzyme. Conclusion: These factors may render TreS II relatively more thermostable among mesophilic trehalose synthases. The thermophilic amino acid residues provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.

Key words： Myxococcus sp.V11 Trehalose synthase Site-directed mutagenesis Thermostability

Received: 27 July 2019 Published: 18 April 2020

ZTFLH:

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Corresponding Authors: Fei WANG E-mail: wangfei179@163.com

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	Xiao-yan ZHAO
	Yun-da CHEN
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	Xiao-yu WU
	Fei WANG
	Jin-yin CHEN

Cite this article:

ZHAO Xiao-yan,CHEN Yun-da,ZHANG Ya-qian,WU Xiao-yu,WANG Fei,CHEN Jin-yin. Site-directed Mutagenesis Improves the Thermostability of Trehalose Synthase TreS II from Myxococcus sp.V11. China Biotechnology, 2020, 40(3): 79-87.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1907049 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/79

Table 1 Oligonucleotides used for site-directed mutagenesis

Fig.1 Sequence alignment of known trehalose synthases Three regions of highly conserved sequence that are shaded in gray boxes. Catalytic residues D202, E244 and E310 are indicated by (*). The metal-binding sites are indicated by (:). Coordinated amino acids with thermal stability are indicated by (.)

Fig.2 Agarose gel electrophoresis of site-directed mutagenesis tresII by overlapping PCR Lane M: λ Hind III marker; Lane 1: pET-29a-tresII(Q3D); Lane 2: pET-29a-tresII(A283R); Lane 3: pET-29a-tresII(W374D); Lane 4: pET-29a-tresII(R449Q); Lane 5: pET-29a-tresII(Y537H); Lane 6: pET-29a-tresII; Lane 7: pET-29a (+)

Fig.3 Analysis of the expression of the mutant enzymes on SDS-PAGE Lane M: Low molecular protein marker; Lane 1: Total protein of E. coli BL21(DE3) harboring pET-29a(+) - treS II (WT); Lane 2: Total protein of E. coli BL21(DE3) harboring pET-29a(+) - treS II (Q3D); Lane 3: Total protein of E. coli BL21(DE3) harboring pET-29a(+) - treS II (A283R); Lane 4 : Total protein of E. coli BL21(DE3) harboring pET-29a(+) - treS II (Y537H); Lane 5: Total protein of E.coli BL21(DE3) harboring pET-29a(+) - treS II (W374D); Lane 6: Total protein of E. coli BL21(DE3) harboring pET-29a(+) - treS II (R449Q); Lane 7: Total protein of E. coli BL21(DE3) harboring pET-29a(+)

Fig.4 Compared with optimum temperature of mutant and wild-type

Fig.5 Compared with optimum pH of recombinant mutant TreS II and wild-type

Fig.6 Compared with specific activity of mutant recombinant enzyme and wild-type

Fig.7 Comparison of thermal stability of WT and thermal stability of mutant enzymes(a) The thermal stability of WT and mutant enzymes on 40℃ (b) The thermal stability of WT and mutant enzymes on 50℃ (c)The thermal stability of WT and mutant enzymes on 60℃


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