Construction of High-yield and High-purity Pectin Methylesterase <i>Aspergillus niger</i> Engineering Strain

HAN Mengmeng; GAI Jinming; JIANG Qingfeng; ZHANG Hui; LI Jie

doi:10.13386/j.issn1002-0306.2020120017

Volume 42 Issue 20

Oct. 2021

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Science and Technology of Food Industry > 2021 > 42(20): 112-118. > DOI: 10.13386/j.issn1002-0306.2020120017

HAN Mengmeng, GAI Jinming, JIANG Qingfeng, et al. Construction of High-yield and High-purity Pectin Methylesterase Aspergillus niger Engineering Strain[J]. Science and Technology of Food Industry, 2021, 42(20): 112−118. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2020120017.

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Construction of High-yield and High-purity Pectin Methylesterase Aspergillus niger Engineering Strain

College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

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Received Date: December 01, 2020
Available Online: August 21, 2021

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Abstract

In order to obtain an engineering strain of Aspergillus niger with high yield and purity of pectin methylesterase and improve the yield of pectin methylesterase, the pectin methylesterase gene pmeA was cloned from the pectinase-producing strain, and Aspergillus niger was transformed by agrobacterium-mediated method, the homozygous recombinant strain TH-2 (glaA::pmeA). The highest enzyme activity in the supernatant on the 9th day of fermentation in the basic fermentation medium reached 467.77 U/mL. At the same time, SDS-PAGE showed that the main background protein α-amylase disappeared, but the acid-stable α-amylase remained. Further the background protein acid-stable α-amylase encoding gene asaA in the recombinant strain TH-2 (glaA::pmeA) was knock out to obtain the homozygous recombinant strain TH-2 (glaA::pmeA∆pyrG∆asaA). After the strain was cultured in a fermentation medium supplemented with 1% ammonium sulfate for 7 days, the main background proteins in the supernatant of the fermentation broth disappeared. However, compared with the homozygous recombinant strain TH-2 (glaA::pmeA), the expression of pectin methylesterase decreased, and the highest enzyme activity was 255.40 U/mL. The optimum temperature of the recombinant pectin methylesteraseis 50 ℃, the suitable temperature range was 40~80 ℃, it could still maintain more than 70% of its enzyme activity at 80 ℃, the suitable pH range was 3.0~5.0, the optimum pH was 4.0. Finally, an engineering strain of Aspergillus niger with high yield and high purity pectin methylesterase was obtained.
- pectin methylesterase,
- Aspergillus niger,
- background protein,
- secretory expression

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References (31)

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