Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis

Mathupala, Saroj P.; Lowe, Susan E.; Podkovyrov, S M; Zeikus, J. G.

doi:10.1016/s0021-9258(19)85426-1

Cited by 79 publications

(9 citation statements)

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“…This indicates that the purified enzyme is a monomer. Vishnu et al [21] described a monomeric enzyme of L amylophilus GV6 of 90 kDa corresponding to a pullulanase Type I. Thermoanaerobacter ethanolicus [21,[26][27][28][29], while the study of an alkaline amylopullulanase from alkalophilic Bacillus sp. KSM-1378 has shown that the two catalytic activities of the enzyme involve two different active sites [30].…”

Section: Sds-page Analysismentioning

confidence: 99%

An Alkalothermophilic Amylopullulanase from the Yeast Clavispora lusitaniae ABS7: Purification, Characterization and Potential Application in Laundry Detergent

et al. 2021

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In the present study, α-amylase and pullulanase from Clavispora lusitaniae ABS7 isolated from wheat seeds were studied. The gel filtration and ion-exchange chromatography revealed the presence of α-amylase and pullulanase activities in the same fraction with yields of 23.88% and 21.11%, respectively. SDS-PAGE showed a single band (75 kDa), which had both α-amylase (independent of Ca2+) and pullulanase (a calcium metalloenzyme) activities. The products of the enzymatic reaction on pullulan were glucose, maltose, and maltotriose, whereas the conversion of starch produced glucose and maltose. The α-amylase and pullulanase had pH optima at 9 and temperature optima at 75 and 80 °C, respectively. After heat treatment at 100 °C for 180 min, the pullulanase retained 42% of its initial activity, while α-amylase maintained only 38.6%. The cations Zn2+, Cu2+, Na+, and Mn2+ increased the α-amylase activity. Other cations Hg2+, Mg2+, and Ca2+ were stimulators of pullulanase. Urea and Tween 80 inhibited both enzymes, whereas EDTA only inhibited pullulanase. In addition, the amylopullulanase retained its activity in the presence of various commercial laundry detergents. The performance of the alcalothermostable enzyme of Clavispora lusitaniae ABS7 qualified it for the industrial use, particularly in detergents, since it had demonstrated an excellent stability and compatibility with the commercial laundry detergents.

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Section: Sds-page Analysismentioning

confidence: 99%

An Alkalothermophilic Amylopullulanase from the Yeast Clavispora lusitaniae ABS7: Purification, Characterization and Potential Application in Laundry Detergent

et al. 2021

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“…The putative SP cleavage site was predicted based on a statistical method (von Heijne 1985). For ease of manipulation, a 2865-bp DNA fragment of the Apu gene, encoding amino acids 75 to 1029 of the mature APU of T. ethanolicus (Mathupala et al 1993;Lin and Leu 2002), was used for rice transformation. The truncated Apu was PCR-amplified and fused upstream of nopaline synthase gene (Nos), aAmy3 or aAmy8 terminators (Sheu et al 1996;Chan and Yu 1998).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Pullulanase activity was determined by a published method (Mathupala et al 1993) with slight modifications. The assay was conducted at 40 °C for 30 min in a reaction mixture (200 ll) containing 1.25% pullulan (w/v), 50 mM acetic acid-NaOH (pH 6.0), 5 mM CaCl 2 , and enzyme preparation (40 ll).…”

Section: Assay Of Pullulanase Activity In Developing Seedsmentioning

confidence: 99%

“…APU from T. ethanolicus 39E, which harbors both a-amylase and pullulanase activities, is capable of hydrolyzing both a-1,4 and a-1,6 bonds of polysaccharides and is heat stable with a catalytic optimum at 90 °C (Mathupala et al 1993;Lin and Leu 2002). The use of transgenic rice seeds containing this thermostable and bi-functional APU would theoretically facilitate liquefaction and saccharification of starch simultaneously at high temperature without the need to add exogenous a-amylase and pullulanase.…”

Section: Introductionmentioning

confidence: 99%

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Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch

et al. 2005

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Overexpression of bacterial-derived starch metabolic enzymes in plant starch storage organs represents a valuable strategy for improving starch quality, bioprocessing and nutritional value. Transgenic rice seeds producing a thermostable and bifunctional starch hydrolase, amylopullulanase (APU) from Thermoanaerobacter ethanolicus 39E, were generated. Starch in these seeds could be hydrolyzed with optimal temperatures between 85 and 95 °C, which resulted in complete conversion of starch into soluble sugars and production of protein-enriched flour within a few hours. By expressing various levels of APU, rice seeds containing reduced amounts of amylose, which is an important factor affecting starch quality, were obtained without a significant impact on grain yield. Elevation in granule-bound pullulanase activity correlates with the reduction of amylose in developing APU-containing rice seeds. APU was found to be localized within amyloplasts and in cell walls, which could be the result of overexpression of APU with a signal peptide. This study establishes novel approaches to alter starch properties, accelerate bioprocessing of starch and production of protein-enriched flour from rice seeds, and could significantly impact the industrial and food uses of cereals.

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“…The catalytic domain-A ((β/α)8-or TIM-barrel) is the most conserved domain in the α-amylase family, and consists of an amino terminal (β/α) 8-barrel structure [36,37]. In the center of this domain, three residues (Asp, Glu, Asp) form the catalytic site as determined by X-ray crystallography [38] and site directed mutagenesis [39]. B-domain protrudes out of the barrel as a longer loop between the strand β3 and helix α3 and succeeded at the C-terminal end by domain C, adopting an antiparallel β-sandwich fold [40].…”

Section: General Features and Activitymentioning

confidence: 99%

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans

Rodríguez-Viera

Alpízar-Pedraza²,

Mancera

et al. 2021

Biology

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Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans.

show abstract

Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis

Cited by 79 publications

References 1 publication

An Alkalothermophilic Amylopullulanase from the Yeast Clavispora lusitaniae ABS7: Purification, Characterization and Potential Application in Laundry Detergent

An Alkalothermophilic Amylopullulanase from the Yeast Clavispora lusitaniae ABS7: Purification, Characterization and Potential Application in Laundry Detergent

Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans

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