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

Abstract

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.

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications

“…Enhancement of B. safensis pullulanase activity by Ca 2+ was in consonant with B. cereus ( Hii et al., 2009 ), Paenibacillus lautus ( Chen et al., 2016 ), edible white mushroom ( Shehata et al., 2016 ), Pyrobaculum calidifontis ( Rehman et al., 2018 ) and Clavispora lusitaniae ABS7 ( Dakhmouche et al., 2021 ), while the inhibition of the activity of the enzyme by Al 3+ , Ca 2+ , Fe 2+ , Pb 2+ , Co 2+ , Cu 2+ , Mg 2+ ions and inhibitor such as EDTA was similar with that of Cryptococcus sp. ( Djekrif et al., 2018 ) which was inhibited by Cu 2+ , Hg 2+ , Zn 2+ , Ni 2+ , Mg 2+ and pullulanase from white edible mushroom ( Shehata et al., 2016 ) strongly inhibited by Hg 2+ , Ag + and Co 2+ .…”

“…Debranching enzymes are capable of catalyzing α-1,6 glycosidic linkages thereby leading to their hydrolysis ( Wu, 2015 ; Dakhmouche et al., 2021 ). Debranching enzymes are classified into indirect and direct branching enzymes.…”

“…Amylo-1,6 glucosidases, indirect debranching enzymes produced by animals and yeast are only capable of hydrolysing a 1,6 - α branch point while pullulanases and isoamylases, classes of direct debranching enzymes found in plants and bacteria have been observed to effectively break α-1,6 glucosidic bond of unmodified substrate ( Hii et al., 2012 ; Zhang et al., 2020 ). Therefore, pullulanase (pullulan α-glucano hydrolase; EC 3.2.1.41) has been highly preferred in industrial processes because of its capability to be involved in saccharification of starch, amylopectin, pullulan and related polysaccharides in combination with other amylolytic enzymes and subsequently yield hydrolysis of α-1, 6 bonds in the polysaccharides with the production of maltose, maltotriose and fructose ( Wu et al., 2015 ; Ma et al., 2015 ; Dakhmouche et al., 2021 ). More so, its usefulness as additives, production of malto syrups, pure glucose and fructose and reduction of dental plaque has drawn more attention of its relevance and requirement ( Zebardast et al., 2017 ; Dakhmouche et al., 2021 ).…”

“…Therefore, pullulanase (pullulan α-glucano hydrolase; EC 3.2.1.41) has been highly preferred in industrial processes because of its capability to be involved in saccharification of starch, amylopectin, pullulan and related polysaccharides in combination with other amylolytic enzymes and subsequently yield hydrolysis of α-1, 6 bonds in the polysaccharides with the production of maltose, maltotriose and fructose ( Wu et al., 2015 ; Ma et al., 2015 ; Dakhmouche et al., 2021 ). More so, its usefulness as additives, production of malto syrups, pure glucose and fructose and reduction of dental plaque has drawn more attention of its relevance and requirement ( Zebardast et al., 2017 ; Dakhmouche et al., 2021 ).…”

“…Pullulanases are widely classified according to their substrate specificities and reaction products ( Ma et al., 2015 ). Most pullulanases that are found in different species of bacteria, yeast, and fungi have been reported to be type II pullulanases ( Kim et al., 1993 ; Dakhmouche et al., 2021 ) while few numbers of type I pullulanases that have been observed are found at gene level ( Suzuki et al., 1991 ). It has however been observed also that, Type I and type II pullulanases work synergistically: Type I acts on linkages in pullan and amylopectin, a branched oligosaccharide yielding maltotriose and oligosaccharides that are branched while Type II pullulanase which has amylase and pullulanase activity releases remaining polysaccharides by cleaving starch glycosidic bonds at α-1,6 and α-1,4 glycosidic linkage ( Zebardast et al., 2017 ).…”