Heterologous Expression, Mutation, Optimizing the Expression Condition and Characterization of Lysostaphin in <i>Kluyveromyces lactis</i>

doi:10.13523/j.cb.20171208

China Biotechnology

2017, Vol. 37

Issue (12): 49-58 DOI: 10.13523/j.cb.20171208

Orginal Article

Heterologous Expression, Mutation, Optimizing the Expression Condition and Characterization of Lysostaphin in Kluyveromyces lactis

Xi WANG¹,Guang-de ZHANG¹,Xi-ming CHEN²,Tong-liang PU^1*(

)

1 School of Life Sciences,LanZhou University, Lanzhou 730000, China
2 Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China

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Abstract

According to the sequence of lysostaphin gene from Staphylococcus simulans and codon bias of Kluyveromyces lactis, the PCR primers were designed to amplify the fragment of lysostaphin gene. The fragment was inserted in pKLAC1, and transformed to K. lactis GG799. The K. lactis GG799/pKLAC1- Lys was cultivated to express Lys. A high expression strain (mu4#) were abtained by using powerful mutagen (N-methy1-N-nitro-N-nitrosoguanidine,NTG) on the recombinant and optimized the expression condition .The fermentation broth of mu4# was purified by Ni-NTA agarose and the enzyme characterization was studied. The result showed that the activity of Lys was approximately 5.2 times (8 000U/L) higher in the mutation. The optimal inoculum dose of the mutant (mu4#) was 40g/L; Galactose and NH₄NO₃ (20g/L) were added in every 24 hours, Lys exhibited optimal expression at pH 7.0~7.5; Furthermore, the Lys enzyme optimal reaction performed at pH 7.0~8.0 and temperature at 37℃. The recombinant Lys was stable below 40℃ and pH between 3.0 and 6.0. Sr²⁺ stimulated its activity whereas Ba²⁺、Ca²⁺、Zn²⁺、Cu²⁺、Mn²⁺、Mg²⁺ inhibited the activities. This research accomplished Lys recombinant expression, yield improvement by chemical mutagenesis in K. lactis and characterization of lysostaphin. These research results provide profound guiding significance for the large-scale production and application of recombinant lysostaphin.

Key words： Kluyveromyces lactis Lysostaphin Recombinant Mutation

Received: 12 June 2017 Published: 16 December 2017

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	Xi WANG
	Guang-de ZHANG
	Xi-ming CHEN
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Xi WANG,Guang-de ZHANG,Xi-ming CHEN,Tong-liang PU. Heterologous Expression, Mutation, Optimizing the Expression Condition and Characterization of Lysostaphin in Kluyveromyces lactis. China Biotechnology, 2017, 37(12): 49-58.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20171208 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I12/49

Table 1 PCR reaction system

Fig.1 Agarose gel electrophoresis of Lys
M:DNA marker; 1: PCR product of Lys

Fig.2 Structure of expression vector pKLAC1-Lys

Fig.3 The enzyme identification of pKLAC1-Lys
M: DNA marker; 1:Expression vector pKLAC1-Lys; 2: Expression vector pKLAC1-Lys digested by XhoⅠand KpnⅠ

Fig.4 Single enzyme digestion of pKLAC1-Lys
M: DNA marker; 1:Expression vector pKLAC1-Lys digested by SacⅡ

Fig.5 The Lys enzyme activity of K. lactis GG799/pKLAC1- Lys 8# during the induction by the galactose

Fig.6 The Lys enzyme activity of mu4# during the induction by the galactose

Fig.7 The growth curve of mu4#

Fig.8 Different initial inoculum dosage of mu4# on Lys activity

Fig.9 Galactose addition of mu4# on Lys activity at different times
a:The addition of galactose at the final concentration of 20g/L in 24h; b: The addition of galactose at the final concentration of 20 g/L in 24h and 48h respectively; c: The addition of galactose at the final concentration of 20 g/L in 24h、48h and 72h respectively; d: The addition of galactose at the final concentration of 20 g/L in 24h、48h、72h and 96h respectivel

Fig.10 Nitrogen source addition of mu4# on Lys activity at different times
a:The addition of NH₄NO₃ at the final concentration of 20g/L in 24h; b: The addition of NH₄NO₃ at the final concentration of 20g/L in 24h and 48h respectively; c: The addition of NH₄NO₃ at the final concentration of 20 g/L in 24h、48h and 72h respectively; d: The addition of NH₄NO₃ at the final concentration of 20 g/L in 24h, 48h, 72h and 96h respectively

Fig.11 The effect of different pH on mu4# Lys activity

Fig.12 SDS-PAGE analysis of mu4# from fermentation broth

Fig.13 The optimum reaction pH of Lys

Fig.14 pH stability of Lys

Fig.15 The optimum reaction temperature of Lys

Fig.16 The thermostability of Lys

Fig.17 The Effect of different divalent mental ions on Lys enzymes activity


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