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Improved saccharification of steam-exploded Pinus radiata on supplementing crude extract of Penicillium sp.

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ABSTRACT

Commercially available enzymes do not contain all the necessary softwood-specific accessory enzymes to obtain high saccharification efficiency. In this work, six saprophytic fungi obtained from Pinus radiata plantation site were screened for the putative softwood-specific accessory enzyme, β-mannanase. A Penicillium sp. was found to produce β-mannanase in both solid (31.6 units/g of dry biomass) and liquid (117 units/g of dry biomass) cultures using locust bean gum as an inducer after 2 weeks of incubation. The saccharification of steam-exploded Pinus radiata was 7.8 % w/w improved when the crude extract of Penicillium sp. was added to a mixture of commercial enzymes.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-014-0212-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


a Crude β-mannanase recovery from solid medium. b Crude β-mannanase recovery from liquid medium. Data shown here are average of quadruplicate measurements with corresponding relative standard deviations. Filled triangles Penicillium sp., empty trianglesCladosporumherbarum, filled squaresOligoporusplacenta, empty squaresLenzitestrabea, filled diamondsCephalosporium sp. and empty diamondsChaetomiumglobosum
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Fig1: a Crude β-mannanase recovery from solid medium. b Crude β-mannanase recovery from liquid medium. Data shown here are average of quadruplicate measurements with corresponding relative standard deviations. Filled triangles Penicillium sp., empty trianglesCladosporumherbarum, filled squaresOligoporusplacenta, empty squaresLenzitestrabea, filled diamondsCephalosporium sp. and empty diamondsChaetomiumglobosum

Mentions: β-Mannanase activity in the supernatant was measured using a partially depolymerized Carob-galactomannan conjugated with Remazol brilliant blue R (Megazyme, Ireland) as the substrate. The substrate stock solution was prepared at 1 % w/v in 0.2 M sodium acetate buffer pH 5.0. The substrate stock and crude enzyme supernatant were incubated separately at 40 °C for 3 h before the assay was performed. In 5 mL capacity glass tubes, 0.5 mL of stock substrate solution was mixed with 0.5 mL of crude enzyme solution and incubated at 40 °C for 2.5 h. The reaction was terminated by the addition of 2.5 mL of ethanol (95 % v/v). After brief vortexing, the absorbance of supernatant was measured at 590 nm. Enzyme activity was determined from a standard calibration curve (R2 = 0.99) of Remazol brilliant blue R solution (0.008–0.00016 μM) prepared in 0.2 M sodium acetate buffer pH 5. One unit of β-mannanase activity was defined as the amount of enzyme which released 0.001 μM of dye under assay conditions. Each assay was performed in quadruplicate and the average value is presented in Fig. 1. β-Glucosidase (E.C. 3.2.1.21) and β-mannosidase (E.C. 3.2.1.25) activity were each measured using 10 mM of standard chromogenic substrates, i.e. p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-mannoside, respectively (Bailey and Nevalainen 1981). One unit of activity was defined as the amount of enzyme which released 1 μM of p-nitrophenol from their respective standard substrates under assay conditions.Fig. 1


Improved saccharification of steam-exploded Pinus radiata on supplementing crude extract of Penicillium sp.
a Crude β-mannanase recovery from solid medium. b Crude β-mannanase recovery from liquid medium. Data shown here are average of quadruplicate measurements with corresponding relative standard deviations. Filled triangles Penicillium sp., empty trianglesCladosporumherbarum, filled squaresOligoporusplacenta, empty squaresLenzitestrabea, filled diamondsCephalosporium sp. and empty diamondsChaetomiumglobosum
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: a Crude β-mannanase recovery from solid medium. b Crude β-mannanase recovery from liquid medium. Data shown here are average of quadruplicate measurements with corresponding relative standard deviations. Filled triangles Penicillium sp., empty trianglesCladosporumherbarum, filled squaresOligoporusplacenta, empty squaresLenzitestrabea, filled diamondsCephalosporium sp. and empty diamondsChaetomiumglobosum
Mentions: β-Mannanase activity in the supernatant was measured using a partially depolymerized Carob-galactomannan conjugated with Remazol brilliant blue R (Megazyme, Ireland) as the substrate. The substrate stock solution was prepared at 1 % w/v in 0.2 M sodium acetate buffer pH 5.0. The substrate stock and crude enzyme supernatant were incubated separately at 40 °C for 3 h before the assay was performed. In 5 mL capacity glass tubes, 0.5 mL of stock substrate solution was mixed with 0.5 mL of crude enzyme solution and incubated at 40 °C for 2.5 h. The reaction was terminated by the addition of 2.5 mL of ethanol (95 % v/v). After brief vortexing, the absorbance of supernatant was measured at 590 nm. Enzyme activity was determined from a standard calibration curve (R2 = 0.99) of Remazol brilliant blue R solution (0.008–0.00016 μM) prepared in 0.2 M sodium acetate buffer pH 5. One unit of β-mannanase activity was defined as the amount of enzyme which released 0.001 μM of dye under assay conditions. Each assay was performed in quadruplicate and the average value is presented in Fig. 1. β-Glucosidase (E.C. 3.2.1.21) and β-mannosidase (E.C. 3.2.1.25) activity were each measured using 10 mM of standard chromogenic substrates, i.e. p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-mannoside, respectively (Bailey and Nevalainen 1981). One unit of activity was defined as the amount of enzyme which released 1 μM of p-nitrophenol from their respective standard substrates under assay conditions.Fig. 1

View Article: PubMed Central

ABSTRACT

Commercially available enzymes do not contain all the necessary softwood-specific accessory enzymes to obtain high saccharification efficiency. In this work, six saprophytic fungi obtained from Pinus radiata plantation site were screened for the putative softwood-specific accessory enzyme, β-mannanase. A Penicillium sp. was found to produce β-mannanase in both solid (31.6 units/g of dry biomass) and liquid (117 units/g of dry biomass) cultures using locust bean gum as an inducer after 2 weeks of incubation. The saccharification of steam-exploded Pinus radiata was 7.8 % w/w improved when the crude extract of Penicillium sp. was added to a mixture of commercial enzymes.

Electronic supplementary material: The online version of this article (doi:10.1007/s13205-014-0212-2) contains supplementary material, which is available to authorized users.

No MeSH data available.