A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin

Wu, Xi; Du, Jun; Zhang, Zhiyun; Fu, Yuejun; Wang, Wenming; Liang, Aihua

doi:10.1007/s00253-018-9327-4

Cited by 11 publications

(6 citation statements)

References 36 publications

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“…The melting temperatures of phytase enzymes generally range from 57°C to 64°C. For example, Tm value was reported as 57°C for Ymphytase WT, 60.3°C for AppA‐WT, 61.5°C for AppA‐M2, 63.8°C for appA‐M5, and 64.1°C for appA‐M6 (Høiberg‐Nielsen et al, 2006; Körfer et al, 2012; Shivange et al, 2014; Shivange et al, 2016; Wang et al, 2018; Xi et al, 2015). Through protein engineering, many variants have shown different levels of thermostability improvement, and the melting temperature can be increased to 73°C (Maurya et al, 2017), including the one engineered in our laboratory (Navone et al, 2021b).…”

Section: Discussionmentioning

confidence: 99%

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Luna‐Flores,

Weng,

Wang

et al. 2023

Biotech & Bioengineering

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Pichia pastoris (Komagataella phaffii) is a fast‐growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co‐express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed‐batch modes. First, the production of phytase was investigated under glucose de‐repression in continuous culture at three dilution factors, 0.5 d−1, 1 d−1, and 1.5 d−1. The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed‐batch modes for growth and phytase production. The kinetic model developed aided to design the glucose‐feeding profile of a fed‐batch culture. Kinetic model simulations under glucose de‐repression and fed‐batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h−1. Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de‐repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5‐fold by methanol induction compared to the expression with glucose alone under de‐repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36‐fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.

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Section: Discussionmentioning

confidence: 99%

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Luna‐Flores,

Weng,

Wang

et al. 2023

Biotech & Bioengineering

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“…Increased H-bonding was observed between N363 and E53. Increased thermostability was obtained as a result of introducing hydrogen bond and N-glycosylation through the increased rigidity of phytase [33].…”

Section: Escherichia Coli Phytasementioning

confidence: 99%

“…The expression system in Pichia pastoris brought about N-glycosylation modification at the N in the consensus sequence N-X-S/T. The reinforcement of the H-bond network in the loop area and N-glycosylation modification enhanced the stability of the phytase structure against trypsin [33].…”

Section: Escherichia Coli Phytasementioning

confidence: 99%

Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry

et al. 2020

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Resistance to high temperature, acidic pH and proteolytic degradation during the pelleting process and in the digestive tract are important features of phytases as animal feed. The integration of insights from structural and in silico analyses into factors affecting thermostability, acid stability, proteolytic stability, catalytic efficiency and specific activity, as well as N-glycosylation, could improve the limitations of marginal stable biocatalysts with trade-offs between stability and activity. Synergistic mutations give additional benefits to single substitutions. Rigidifying the flexible loops or inter-molecular interactions by reinforcing non-bonded interactions or disulfide bonds, based on structural and roof mean square fluctuation (RMSF) analyses, are contributing factors to thermostability. Acid stability is normally achieved by targeting the vicinity residue at the active site or at the neighboring active site loop or the pocket edge adjacent to the active site. Extending the positively charged surface, altering protease cleavage sites and reducing the affinity of protease towards phytase are among the reported contributing factors to improving proteolytic stability. Remodeling the active site and removing steric hindrance could enhance phytase activity. N-glycosylation conferred improved thermostability, proteases degradation and pH activity. Hence, the integration of structural and computational biology paves the way to phytase tailoring to overcome the limitations of marginally stable phytases to be used in animal feeds.

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“…-Íons ativadores da atividade da fitase em duas diferentes (SULEIMANOVA et al, 2015;EMMENES et al, 2018;WANG et al, 2018;VILLAMIZAR, et al, 2019a;VERARDI et al, 2019).…”

Section: Lista De Figurasunclassified

“…O ácido fítico, um grupo complexo de componentes naturais, encontrado em plantas e no solo (ROSTAMI;GIRI, 2013;MITTAL et al, 2013), é considerado a maior forma de armazenamento do fósforo nos alimentos vegetais (cerca de 50 a 80%), especialmente os cereais e leguminosas (FARHADI et al, 2019;SHARIDEH et al, 2019;VASUDEVAN et al, 2019), principais ingredientes das rações para aves e suínos (WANG et al, 2018;VASUDEVAN et al, 2019).…”

Section: Pmsfunclassified

Clonagem, expressão e caracterização de fitase do fungo termofílico >i<Myceliophthora thermophila>/i<

Ferreira¹

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Fitases são enzimas que hidrolisam o ácido fítico, substâncias orgânicas complexas amplamente encontradas na natureza, principalmente em cereais e leguminosas. Nesses alimentos vegetais, o fitato é a principal fonte de fósforo, correspondendo entre cinquenta a oitenta por cento do conteúdo de fósforo orgânico. Entretanto, o fósforo do fitato é muito pouco utilizado por humanos e outros animais monogástricos, como suínos e aves, devido à ausência ou insuficiência das enzimas que o degradam. Assim, rações contêm o fósforo orgânico do fitato e fósforo inorgânico adicionado, que é um mineral caro e não renovável. Em áreas de criação intensiva o excesso de fósforo é excretado no ambiente, causando problemas, tais como a eutrofização. Devido aos problemas ambientais e ao alto custo do fósforo inorgânico, a suplementação de enzimas, principalmente fitases, tem ganhado destaque, entre elas, as enzimas fúngicas, devido a sua estabilidade a diferentes temperaturas e pH. Além disso, técnicas de engenharia genética permitem a transferência de genes entre espécies, resultando em muitos benefícios, principalmente em relação ao aumento e melhoramento na produção de enzimas industriais. O objetivo deste trabalho foi realizar a clonagem, expressão heteróloga, purificação e caracterização bioquímica da enzima fitase (MtPhy) obtida a partir do fungo termofílico M. thermophila. O gene que codifica a enzima foi clonado no vetor pEXPYR e heterologamente expresso em A. nidulans. A proteína recombinante foi purificada e teve sua identidade confirmada por espectrometria de massas. Nas análises bioquímicas, apresentou pH ótimo de 5,0 e temperatura ótima de 60°C e manteve praticamente 100% de sua atividade a 50°C por 2 horas e pela análise de dicroísmo circular e ThermoFluor, apresentou desenovelamento de sua estrutura nas temperaturas de 63 e 67°C, respectivamente. Demonstrou atividade aumentada na presença de CaCl 2 e MgCl 2 e foi fortemente inibida por FeCl 3 , CuSO 3 e SDS. Sua atividade específica (954,8 U/mg) utilizando fitato de sódio foi bastante superior a de outras fitases descritas na literatura. Demonstrou-se efetiva na liberação do fósforo em rações e extremamente resistente às proteases, tripsina e pepsina, presentes no trato digestório de animais monogástricos, demonstrando dessa forma, seu elevado potencial para aplicação industrial. Palavras-chave: Fitases. Fungos termofílicos. Myceliophtora thermophila.

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A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin

Cited by 11 publications

References 36 publications

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Improving phytase production in Pichia pastoris fermentations through de‐repression and methanol induction optimization

Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry

Clonagem, expressão e caracterização de fitase do fungo termofílico >i<Myceliophthora thermophila>/i<

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