Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch

Martı́nez, T.F.; Alarcón, Francisco Javier; López, Manuel Díaz; Moyano, Francisco Javier

doi:10.1002/1522-2683(20000801)21:14<2940::aid-elps2940>3.0.co;2-s

Cited by 27 publications

(23 citation statements)

References 13 publications

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“…Some of the methods described in the literature recommended this procedure [7,8,14]. According to our results, it is highly desirable that both enzyme samples and MMM are carried out in the same gel, overcoming differences in migration produced when MMM are carried out in a parallel electrophoresis in absence of substrate.…”

Section: Discussionmentioning

confidence: 93%

“…Moreover, small differences in substrate concentration among gels, which could affect Rf, can be overcome if MMM are included in a single gel. It can be difficult to visualize MMM when they are included in substrate-SDS-PAGE, but they can be revealed in starch-SDS-PAGE by cutting a slice of the gel containing MMM proteins as explained elsewhere [7]. Protein-SDS-PAGE also allows the revealing of MMM by cutting a gel slice containing standard proteins, although some difficulties can be encountered, especially when protein substrates are included at concentrations over 0.2%.…”

Section: Discussionmentioning

confidence: 99%

“…That was supported by the observation that the enzymes hydrolysed the substrate during electrophoretic migration. However, the hydrolysis of the substrates during enzyme migration is widely known, and diverse procedures have been proposed to overcome such effect [7,8].…”

Section: Discussionmentioning

confidence: 99%

“…Unfortunately for many enzymes reaction with SDS results in inactivation, and a renaturation step is required after electrophoresis [1]. However, there are reports of SDS-tolerant enzymes [2][3][4][5][6][7], which can be separated in SDS-PAGE without a renaturation step.…”

Section: Introductionmentioning

confidence: 99%

“…Since it was first proposed by Heussen and Dowdle [8], electrophoretic separation of enzymes under denaturing conditions in the presence of high-molecular-mass substrates incorporated in the polyacrylamide (PA) matrix has been used with starch [7], gelatin [8][9][10], and collagen [11]. These methods are based on the sensitivity of substrates that, when trapped into the PA matrix at the time of casting, are retained during subsequent electrophoresis of enzyme samples, so providing in situ substrates for previously separated active fractions.…”

Section: Introductionmentioning

confidence: 99%

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Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Martı́nez

Newbold²,

Wallace³

et al. 2002

Electrophoresis

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Substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has become a popular procedure for the separation and identification of active fractions present in enzyme mixtures due to its relative simplicity. Procedures including high-molecular-mass substrates within the gel, such as starch for identification of amylase activity, and protein substrates, including gelatin, casein, and collagen, for revealing protease activity, have been described. SDS-PAGE separation under denaturing conditions is dependent on the molecular mass of the proteins and on the effective pore size of the gels, the last factor being affected by the inclusion of high-molecular-mass substrates into the polyacrylamide matrix. In order to quantify the effect of the addition of increasing concentrations of such substrates on protein migration, starch, gelatin, and casein were included in gels in which polyacrylamide concentration was kept constant. High-molecular-mass substrates decreased migration of proteins ranging from 6.5 to 205 kDa, although the migration pattern, and thereby the accuracy of the assignation of relative molecular masses to proteins separated on those gels, was practically unaffected. The substitution of glycine, as the carrying ion, by Tricine in denaturing electrophoresis buffer systems resulted in an improvement of the migration of proteins in substrate-containing gels. Results suggested that zymograms including substrates remain a valuable procedure for the separation and the relative molecular mass assignation of active enzyme fractions.

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Martı́nez

Newbold²,

Wallace³

et al. 2002

Electrophoresis

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Plant cell calcium‐rich environment enhances thermostability of recombinantly produced α‐amylase from the hyperthermophilic bacterium Thermotoga maritime

Santa-María

Chou

Yencho

et al. 2009

Biotech & Bioengineering

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In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

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Starch self‐processing in transgenic sweet potato roots expressing a hyperthermophilic α‐amylase

Santa-María

Yencho

Haigler

et al. 2011

Biotechnology Progress

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Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.

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Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch

Cited by 27 publications

References 13 publications

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Plant cell calcium‐rich environment enhances thermostability of recombinantly produced α‐amylase from the hyperthermophilic bacterium Thermotoga maritime

Starch self‐processing in transgenic sweet potato roots expressing a hyperthermophilic α‐amylase

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