High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme

Zhou, Chen; Yan, Qiaojuan; Jiang, Zhengqiang; Yu, Li; Li, Yuchen

doi:10.1016/j.pep.2015.02.015

Cited by 27 publications

(14 citation statements)

References 28 publications

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“…pastoris . Their extracellular enzymatic activities, as determined with the PNPG ( p -nitrophenyl- α -D-galactopyranoside) method [16], were reported to be 1.5 U/ml [17], 111 U/ml [18] and 240 U/ml [19], respectively, upon a 96 h methanol induction. When an α -galactosidase gene from Penicillium janczewskii zalesk was mutated by error-prone PCR and DNA shuffling, and then expressed in P .…”

Section: Introductionmentioning

confidence: 99%

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

et al. 2016

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α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.

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Section: Introductionmentioning

confidence: 99%

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

et al. 2016

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“…α‐Galactosidases from GH family 27 are typically classified into one group and exhibit enzymatic activities on short oligosaccharides and polymeric substrates . By contrast, GH family 36 α‐galactosidases present high specificity toward small oligosaccharides .…”

Section: Discussionmentioning

confidence: 99%

“…α‐Galactosidases are a group of exoglycosidases catalyzing the cleavage of terminal α‐1,6‐linked galactosyl residues of α‐galactosides, including RFOs, galacto (gluco)‐mannans, and galactolipids . α‐Galactosidases are involved in various important applications.…”

Section: Introductionmentioning

confidence: 99%

Purification of thermostable α‐galactosidase from Irpex lacteus and its use for hydrolysis of oligosaccharides

Guo

Song

Qiu

et al. 2016

J. Basic Microbiol.

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A monomeric α-galactosidase (ILGI) from the mushroom Irpex lacteus was purified 94.19-fold to electrophoretic homogeneity. ILGI exhibited a specific activity of 18.36 U mg(-1) and demonstrated a molecular mass of 60 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ILGI was optimally active at 80 °C and pH 5.0, and it was stable over a temperature range of 4-70 °C and a wide pH range of 2.0-12.0. ILGI was completely inactivated by Ag(+) and Hg(2+) ions and N-bromosuccinimide (NBS). Moreover, ILGI exhibited good resistance to proteases. Galactose acted as a noncompetitive inhibitor with Ki and Kis of 3.34 and 0.29 mM, respectively. The α-galactosidase presented a broad substrate specificity, which included p-nitrophenyl α-D-galactopyranoside (pNPGal), melibiose, stachyose, and raffinose with Km values of 1.27, 3.24, 7.1, and 22.12 mM, correspondingly. ILGI exhibited efficient and complete hydrolysis to raffinose and stachyose. The aforementioned features of this enzyme suggest its potential value in food and feed industries.

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“…The enzyme was used for the production of GOS using 36% (w/v) lactose resulting in 50.5% GOS on dry weight basis. Immobilization of β‐galactosidase on magnetic nanoparticles are mainly for feasible recovery of the enzyme and reuse for economic considerations (Chen, Qiaojuan, Zhengqiang, Yu, & Yuchen, ). Peirce et al () reported coimmobilization of β‐galactosidase from Aspergillus oryzae and lipase B from Candida antarctica , where the combi‐catalyst provided optimum conditions for lipase immobilization on octyl‐agarose.…”

Section: Cell and Enzyme Immobilization And Process Optimizationmentioning

confidence: 99%

“…Immobilization of b-galactosidase on magnetic nanoparticles are mainly for feasible recovery of the enzyme and reuse for economic considerations (Chen, Qiaojuan, Zhengqiang, Yu, & Yuchen, 2015). Peirce et al to the traditional methodology of immobilization using glutaraldehyde which is not food grade.…”

Section: Recombinantmentioning

confidence: 99%

β-galactosidase: Biotechnological applications in food processing

2018

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β‐Galactosidases produced by microorganisms are being used in food technology for hydrolysis of lactose in milk and milk by‐products. The enzyme has attracted much attention in view of lactose intolerance in human population and due to importance of milk in human diet. β‐Galactosidase hydrolyze β‐galactopyranosides, that is, lactose, and form a range of trans‐galactosylation products or galactooligosaccharides (GOS) capable of providing several health benefits as prebiotics. In addition, the enzyme also finds applications in production of lactose based sweeteners from high lactose containing effluents of cheese manufacturing industries. Currently, the enzyme is mainly obtained from Aspergillus niger and Kluyveromyces lactis, however, they are thermolabile with optimum pH between 4.0 and 6.0 accompanied by low chemical resistance and high product inhibition properties. Thermostable enzymes are suitable to perform the hydrolytic process at relatively high temperatures, minimizing pathogen contamination, psychrophilic β‐galactosidases are preferred for treatment of milk under refrigerated conditions (<15°C), without heating and maintenance of temperature. Lactic acid bacteria (LAB) are a potential source of food grade enzymes and other metabolites. Although reports on the use of β‐galactosidase of LAB in pure form is reported for hydrolysis of lactose in milk and production of fermented milk products for reduced lactose content, methods of immobilization for use in continuous bioreactors are less explored. Recombinant DNA technology has attained much significance to reveal molecular aspects of enzyme catalysis, protein structure, gene organization for expression, and enhancement of thermophilic, psychrophilic, and mesophilic β‐galactosidases. Heterologous expression of extremozymes with β‐galactosidase would help in overcoming the limitations faced within the bioprocess technology. Practical applications Lactic acid bacteria (LAB) producing extracellular cell bound β‐galactosidase have been isolated and enzyme production was carried out using modified MRS medium incorporating lactose as enzyme inducer. Process for growing probiotic lactic acid bacteria for β‐galactosidase from fermented milk products has been patented (Indian Patent No. 247904). Thermostable β‐galactosidases were also extracted from Kluyveromyces lactis a yeast, isolated from curd samples and Bacillus sp. MTCC 864. An Indian patent has been granted for a simple, cost effective perforated two compartment immobilized enzyme bioreactor was designed and fabricated for lactose hydrolysis (Indian Patent No. 264609). The bioreactor can be operated continuously at temperature between 5.0 and 75.0°C, employing different immobilized enzyme preparations for different operational temperatures. β‐galactosidase of cell confined LAB and yeast have been immobilized in agarose and galactooligosaccharides (GOS) production was observed at 40% lactose concentration in 0.05M phosphate buffer at pH 6.5. Immobilized bioreactor preparation could be used for lactos...

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High-level expression of a novel α-galactosidase gene from Rhizomucor miehei in Pichia pastoris and characterization of the recombinant enyzme

Cited by 27 publications

References 28 publications

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

Purification of thermostable α‐galactosidase from Irpex lacteus and its use for hydrolysis of oligosaccharides

β-galactosidase: Biotechnological applications in food processing

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