Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics

Maurya, Anay Kumar; Parashar, Deepak; Satyanarayana, T.

doi:10.1016/j.ijbiomac.2016.09.102

Cited by 19 publications

(9 citation statements)

References 40 publications

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“…From the growth profile, it was observed that the change in pH or temperature from optimum values had a negative effect on both cell growth and Ba-Gt-amy production, as reported earlier by Várnai et al (2014) that optimum cultivation conditions favor recombinant protein expression. In contrast, increase in the yield of heterologous protein on altering temperature or pH beyond the optimum was observed; this could be because it facilitates folding and assembly of heterologous protein due to decrease in the protease activity of P. pastoris (Dragosits et al, 2009; Maurya et al, 2017). …”

Section: Resultsmentioning

confidence: 96%

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

Parashar

Satyanarayana

2017

Front. Microbiol.

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Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2 of 40 min at 70°C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.

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Section: Resultsmentioning

confidence: 96%

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

Parashar

Satyanarayana

2017

Front. Microbiol.

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“…The crude DPE enzyme was concentrated 5 times by ultrafiltration by using the membrane of molecular weight cutoffs of 10 kDa operated at 4 °C. The crude DPE was checked by SDS‐PAGE and Western Blotting analyzes …”

Section: Methodsmentioning

confidence: 99%

“…The crude DPE enzyme was concentrated 5 times by ultrafiltration by using the membrane of molecular weight cutoffs of 10 kDa operated at 4 ∘ C. The crude DPE was checked by SDS-PAGE and Western Blotting analyzes. 31 DPE enzyme immobilization onto anion exchange resin was done as follows. Anion exchange resin D301 (Success Resi Corporation, Jiangsu, China) was immersed and washed three times in deionized water until there was no obvious pigment detected in the eluate.…”

Section: Preparation Of Crude Purified and Immobilized Enzymementioning

confidence: 99%

D‐psicose 3‐epimerase secretory overexpression, immobilization, and d‐psicose biotransformation, separation and crystallization

Lin

Guo

et al. 2017

J of Chemical Tech & Biotech

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BACKGROUND D‐psicose is a rare sugar and exists in extremely small quantities in nature. It has important physiological functions and is allowed to be used as an ingredient in foods and dietary supplements. The aim of this study is to develop the biotransformation, separation and purification methods for highly efficient mass production of d‐psicose. RESULTS First, the gene of d‐psicose 3‐epimerase (DPE) from Ruminococcus sp. was cloned and overexpressed in the food‐grade microorganism Bacillus pumilus, and the recombinant protein was soluble, bioactive, and secretory overexpressed at a high level. The substrate specificity and metal ion effects of DPE were investigated. The recombinant DPE was immobilized onto anion exchange resin matrix, and the enzymatic properties and rounds of reuses of the immobilized DPE were investigated. Then, d‐psicose was produced using the immobilized enzyme, separated by simulated moving bed chromatography (SMB), and finally purified by crystallization. The purity of the d‐psicose crystal reached 99.1%. CONCLUSION Food grade high purity d‐psicose can be efficiently produced and purified using the methods developed in this research, which can be easily scaled up for industrial‐scale mass production. © 2017 Society of Chemical Industry

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“…MAURYA; PARASHAR; SATYANARAYANA, 2017;VASUDEVAN et al, 2019;BENINCASA et al, 2019), além de manganês (BERWAL;CHUGH, 2018;BENINCASA et al, 2019) ;STEIN et al, 2019;CHEN et al, 2019). CHUGH, 2018;VASUDEVAN, et al, 2019).…”

Section: Efeitos Prejudiciais Do Fitatounclassified

“…Para assegurar os requerimentos nutricionais diários de fósforo dos animais, principalmente em áreas de criação intensiva de suínos e frangos, as rações a base de soja e milho vêm sendo enriquecidas com fósforo inorgânico, um mineral não renovável e de alto custo (AARHUS UNIVERSITET 2017;VIEIRA et al, 2019;CHEN et al, 2019). Consequentemente, o fósforo não absorvido ligado ao fitato, bem como o excesso de fósforo inorgânico adicionado às rações, são excretados nas fezes desses animais, causando sérios problemas (ZAREI, 2007;MAURYA;PARASHAR;SATYANARAYANA, 2017), uma vez que no solo encontram-se microrganismos que podem degradar o fitato e, desta maneira, aumentar a liberação de fósforo no ambiente (ZHAO et al, 2007;DAHIYA, 2016).…”

Section: Problemas Ambientaisunclassified

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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Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics

Cited by 19 publications

References 40 publications

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

D‐psicose 3‐epimerase secretory overexpression, immobilization, and d‐psicose biotransformation, separation and crystallization

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

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