Characterization and Multiple Applications of a Highly Thermostable and Ca2+-Independent Amylopullulanase of the Extreme Thermophile Geobacillus thermoleovorans

Abstract

The amylopullulanase of Geobacillus thermoleovorans NP33 (apu105) is Ca(2+)-independent with a molecular mass of 105 kDa and optimum activity at 80 °C and pH 7.0. The apu105 is extremely thermostable with T 1/2 of 7.8 h at 90 °C and hydrolyzes starch, pullulan, and malto-oligosaccharides, but not panose and cyclodextrins. The low K m values of apu105 (starch, pullulan, amylose, and amylopectin) indicates higher affinity of apu105 than others. The action of the enzyme on mixed substrates (starch and pullulan) confirmed the presence of only one active site for cleaving both α-1,4- and α-1,6- glycosidic linkages. The raw starches are efficiently hydrolyzed into glucose, maltose, and malto-oligosaccharides. Two-step starch saccharification involving pretreatment with apu105 followed by glucoamylase enhanced glucose yield. The supplementation of whole wheat dough with apu105 markedly enhanced the loaf volume, shelf-life, and the texture of bread. The enzyme is compatible with detergents and useful in desizing of cotton fabrics.

Revisiting the scope and applications of food enzymes from extremophiles

“…This enzyme had optimum activity at 80°C, was thermostable (half‐life of 7.8 hr at 90°C), able to degrade raw starch, and did not require Ca 2+ as cofactor. Moreover, incorporation of this enzyme in dough improved loaf volume, shelf life, and the texture of bread (Nisha & Satyanarayana, 2014). Therefore, the application of this enzyme eliminates the use of glucoamylase and α‐amylases, shortens the process, thereby having the potential to reduce saccharification cost.…”

Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications

“…The amylopullulanase of G. thermoleovorans NP33 (apu105) had a molecular mass of 105 kDa, while the molecular mass of G. stearothermophilus L14 amylopullulanase was 100 kDa. The amylopullulanase of G. thermoleovorans NP33 (apu105) [52] is highly thermostable with optimum activity at 80 o C and pH 7.0, while 65 o C and pH 5.5 are degrades exogenous substrate resulting in low-molecular weight products, which enter the cells and induce enzyme synthesis. There is no requirement for additional trace elements [34].…”

“…The G. thermoleovorans NP33 amylopullulanase pretreated raw starches have been saccharified to a greater extent by the glucoamylase in comparison to the starch saccharification achieved with the α-amylase pre-treated starches and glucoamylase alone [52]. An improved thermostability and catalytic efficiency achieved with the protein engineering effort via C-terminal truncation enabled efficient use of the enzyme in starch saccharification [3].…”

“…The debranching action of amylopullulanase enables the hydrolysis of the branched maltodextrins, thereby eliminating the stickiness of the α-amylase-treated bread and other bakery products [100]. The supplementation of whole wheat dough with the G. thermoleovorans NP33 amylopullulanase markedly improves the quality of the bread by enhancing the shelf-life, texture of bread and the loaf volume [52]. Moreover, maltooligosaccharides generated in the bread by the enzyme action will act as prebiotics in ameliorating human health.…”

Biochemical Characterization of a Novel Thermostable Type I Pullulanase Produced Recombinantly in Bacillus subtilis

“…The enzymes from Geobacillus sp. have been classified as thermophilic and employed for several industrial uses, including amylopullulanases from Geobacillus thermoleovorans NP33, type I pullulanase from Geobacillus thermoleovorans US105 and type II pullulanase from Bacillus stearothermophilus G‐82 . Although, the Geobacillus species is well‐known for their ability to produce thermophilic enzymes, only a few reports about pullulanases from G eobacillus species, and no report about pullulanases from Geobacillus kaustophilus (including the wild and recombinant strain) exists.…”