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Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

Xu X, Zhang Y, Meng Q, Meng K, Zhang W, Zhou X, Luo H, Chen R, Yang P, Yao B - PLoS ONE (2013)

Bottom Line: The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds.Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.

Methodology/principal findings: The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.

Conclusion/significance: This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Show MeSH
Property comparison of recombinant β-mananases expressed in maize (MAN5AS) and P. pastoris (MAN5A-SST).a) pH-dependent activity profiles of MAN5A-SST and MAN5AS at 65°C. b) pH stability of MAN5A-SST and MAN5AS activities at 37°C for 1 h. c) Temperature-dependent activity profiles of MAN5A-SST and MAN5AS at pH 1.5. d) Thermostability of MAN5A-SST (dashed line) and MAN5AS (solid line) at 60°C (diamond) or 90°C (square) at pH 1.5. Error bars represent the standard deviation of triplicate measurements.
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pone-0056146-g005: Property comparison of recombinant β-mananases expressed in maize (MAN5AS) and P. pastoris (MAN5A-SST).a) pH-dependent activity profiles of MAN5A-SST and MAN5AS at 65°C. b) pH stability of MAN5A-SST and MAN5AS activities at 37°C for 1 h. c) Temperature-dependent activity profiles of MAN5A-SST and MAN5AS at pH 1.5. d) Thermostability of MAN5A-SST (dashed line) and MAN5AS (solid line) at 60°C (diamond) or 90°C (square) at pH 1.5. Error bars represent the standard deviation of triplicate measurements.

Mentions: The crude proteins of transgenic BC2 seeds and P. pastoris fermentation broth were characterized and compared (Figure 5). Both crude enzymes had pH optimum at 4.0, remained active at 1.0–6.0, and retained stable at pH 1.0–11.0. The temperature optimum of MAN5AS was 90°C, 10°C higher than that of MAN5A-SST. Thermostability of MAN5AS and MAN5A-SST were similar, retaining ∼80% activity at 60°C for 60 min and completely inactivated at 90°C for 20 min.


Overexpression of a fungal β-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization.

Xu X, Zhang Y, Meng Q, Meng K, Zhang W, Zhou X, Luo H, Chen R, Yang P, Yao B - PLoS ONE (2013)

Property comparison of recombinant β-mananases expressed in maize (MAN5AS) and P. pastoris (MAN5A-SST).a) pH-dependent activity profiles of MAN5A-SST and MAN5AS at 65°C. b) pH stability of MAN5A-SST and MAN5AS activities at 37°C for 1 h. c) Temperature-dependent activity profiles of MAN5A-SST and MAN5AS at pH 1.5. d) Thermostability of MAN5A-SST (dashed line) and MAN5AS (solid line) at 60°C (diamond) or 90°C (square) at pH 1.5. Error bars represent the standard deviation of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569411&req=5

pone-0056146-g005: Property comparison of recombinant β-mananases expressed in maize (MAN5AS) and P. pastoris (MAN5A-SST).a) pH-dependent activity profiles of MAN5A-SST and MAN5AS at 65°C. b) pH stability of MAN5A-SST and MAN5AS activities at 37°C for 1 h. c) Temperature-dependent activity profiles of MAN5A-SST and MAN5AS at pH 1.5. d) Thermostability of MAN5A-SST (dashed line) and MAN5AS (solid line) at 60°C (diamond) or 90°C (square) at pH 1.5. Error bars represent the standard deviation of triplicate measurements.
Mentions: The crude proteins of transgenic BC2 seeds and P. pastoris fermentation broth were characterized and compared (Figure 5). Both crude enzymes had pH optimum at 4.0, remained active at 1.0–6.0, and retained stable at pH 1.0–11.0. The temperature optimum of MAN5AS was 90°C, 10°C higher than that of MAN5A-SST. Thermostability of MAN5AS and MAN5A-SST were similar, retaining ∼80% activity at 60°C for 60 min and completely inactivated at 90°C for 20 min.

Bottom Line: The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds.Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.

ABSTRACT

Background: Mannans and heteromannans are widespread in plants cell walls and are well-known as anti-nutritional factors in animal feed. To remove these factors, it is common practice to incorporate endo-β-mannanase into feed for efficient nutrition absorption. The objective of this study was to overexpress a β-mannanase gene directly in maize, the main ingredient of animal feed, to simplify the process of feed production.

Methodology/principal findings: The man5A gene encoding an excellent β-mannanase from acidophilic Bispora sp. MEY-1 was selected for heterologous overexpression. Expression of the modified gene (man5As) was driven by the embryo-specific promoter ZM-leg1A, and the transgene was transferred to three generations by backcrossing with commercial inbred Zheng58. Its exogenous integration into the maize embryonic genome and tissue specific expression in seeds were confirmed by PCR and Southern blot and Western blot analysis, respectively. Transgenic plants at BC3 generation showed agronomic traits statistically similar to Zheng58 except for less plant height (154.0 cm vs 158.3 cm). The expression level of MAN5AS reached up to 26,860 units per kilogram of maize seeds. Compared with its counterpart produced in Pichia pastoris, seed-derived MAN5AS had higher temperature optimum (90°C), and remained more β-mannanase activities after pelleting at 80°C, 100°C or 120°C.

Conclusion/significance: This study shows the genetically stable overexpression of a fungal β-mannanase in maize and offers an effective and economic approach for transgene containment in maize for direct utilization without any purification or supplementation procedures.

Show MeSH