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Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris / Mutação do gene da xilanase por pcr propensa a erros e expressão em Pichia pastoris
Li, Shuli; Huang, Weiwei; Chang, Juan; Wang, Ping; Yin, Qingqiang; Liu, Chaoqi; Wang, Erzhu; Lu, Fushan.
  • Li, Shuli; Henan Agricultural University. Zhengzhou. CN
  • Huang, Weiwei; Henan Agricultural University. Zhengzhou. CN
  • Chang, Juan; Henan Agricultural University. Zhengzhou. CN
  • Wang, Ping; Henan Agricultural University. Zhengzhou. CN
  • Yin, Qingqiang; Henan Agricultural University. Zhengzhou. CN
  • Liu, Chaoqi; Henan Agricultural University. Zhengzhou. CN
  • Wang, Erzhu; Henan Delin Biological Product. Xinxiang. CN
  • Lu, Fushan; Henan Engineering and Technology Research Center of Feed Microbes. Zhoukou. CN
Biosci. j. (Online) ; 34(3): 769-777, mai/jun. 2018. tab, ilus, graf
Article in English | LILACS | ID: biblio-966998
ABSTRACT
Xylanase can hydrolyze xylan for reducing its anti-nutritional impact and improving nutrient availability, so obtaining suitable xylanase to degrade xylan is essential. Error-prone PCR and gene transformation were used in this study to obtain the ideal xylanase for degrading xylan effectively. The result showed that one mutant xylanase gene with high xylanase expression was obtained. After the mutant xylanase gene was connected with pGAPZA and transformed into Pichia pastoris (P. pastoris), the recombinant P. pastoris with mutant gene was found to produce higher xylanase activity (0.1480 U/mL) than that with the native xylanase gene (0.1360 U/mL) after 12 h incubation (p<0.05). The optimal temperature and pH of xylanase expressed by native and mutant genes were the same, i.e. 40°C and 5.50 (p<0.05). In addition, adding 0.2% Tween 80 during recombinant P. pastoris incubation could significantly increase xylanase yield by about 30-35% (p<0.05). The mutant xylanase could significantly increase xylose yield from wheat meal more than the native xylanase (p<0.05).
RESUMO
A xilanase pode hidrolisar o xilano para reduzir seu impacto antinutricional e melhorar a disponibilidade de nutrientes, portanto, obter xilanase adequada para degradar o xilano é essencial. A PCR propensa a erros e a transformação genética foram utilizadas neste estudo para obter a xilanase ideal para degradar eficazmente a xilana. O resultado mostrou que um gene mutante de xilanase com alta expressão de xilanase foi obtido. Depois que o gene mutante da xilanase foi conectado ao pGAPZA e transformado em Pichia pastoris (P. pastoris), o recombinante P. pastoris com o gene mutante produziu maior atividade de xilanase (0,1480 U / mL) do que com o gene nativo da xilanase (0,1360 U / mL) após 12 h de incubação (p <0,05). A temperatura e o pH ótimos da xilanase expressa pelos genes nativos e mutantes foram os mesmos, ou seja, 40 ºC e 5,50 (p <0,05). Além disso, a adição de Tween 80 a 0,2% durante a incubação de P. pastoris recombinante poderia aumentar significativamente o rendimento de xilanase em cerca de 30-35% (p <0,05). A xilanase mutante poderia aumentar significativamente o rendimento de xilose da farinha de trigo mais do que a xilanase nativa (p <0,05).
Subject(s)


Full text: Available Index: LILACS (Americas) Main subject: Xylans / Polymerase Chain Reaction Language: English Journal: Biosci. j. (Online) Journal subject: Agricultura / Disciplinas das Ciˆncias Biol¢gicas / Pesquisa Interdisciplinar Year: 2018 Type: Article Affiliation country: China Institution/Affiliation country: Henan Agricultural University/CN / Henan Delin Biological Product/CN / Henan Engineering and Technology Research Center of Feed Microbes/CN

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Full text: Available Index: LILACS (Americas) Main subject: Xylans / Polymerase Chain Reaction Language: English Journal: Biosci. j. (Online) Journal subject: Agricultura / Disciplinas das Ciˆncias Biol¢gicas / Pesquisa Interdisciplinar Year: 2018 Type: Article Affiliation country: China Institution/Affiliation country: Henan Agricultural University/CN / Henan Delin Biological Product/CN / Henan Engineering and Technology Research Center of Feed Microbes/CN