A novel cold‐adapted pullulanase from Exiguobacterium sp. SH3: Production optimization, purification, and characterization

Abstract

Cold‐adapted enzymes, elaborated by psychrophiles and psychrotrophs, seem to have potential for current and future applications. In the current study, pullulanase production by the cold‐adapted Exiguobacterium sp. SH3 was investigated. The Plackett–Burman design and the response surface methodology were applied to identify and optimize the significant variables affecting the pullulanase production of Exiguobacterium sp. SH3. The results showed that temperature, time, and CaCl2 concentration were significant variables while shaking, starch, yeast extract, tryptone, pH, MnCl2, MgCl2, and KH2PO4 were not significant. Using statistical analyses and optimizations, the pullulanase production was significantly elevated from 200 ± 18 to 950 ± 27 U/mL (4.75 times) as compared to non‐optimized conditions. A pullulanase of about 70 kDa, designated as Pul‐SH3, was purified 16.2‐folds from the optimized culture, and identified to be an amylopullulanase. The Km and Vmax of the enzyme were 0.069 mg/mL and 967 U/mL, respectively. The optimum pH and temperature for maximum activity of Pul‐SH3 were 7.5 and 30°C, respectively. As a cold‐adapted enzyme, Pul‐SH3 retained 23% of the maximum activity at 0°C. The biochemical characteristics and N‐terminal amino acid sequence of Pul‐SH3 suggest that the enzyme is a novel cold‐adapted amylopullulanase with remarkably high specific activity at moderate ambient temperature.

Structural and biochemical properties of a novel pullulanase of Paenibacillus lautus DSM 3035

“…In the wheat fermentation industry, pullulanase combined with fungal α‐amylase or glucoamylase can produce low carbohydrate “light beer” . Based on the substrate specificity and end product, pullulanases are classified into five groups: (i) pullulanase type I (EC 3.2.1.41), which specifically hydrolyzes α‐(1,6)‐glycosidic linkages in pullulan or branched oligosaccharides to produce maltotriose; (ii) pullulanase type II (amylopullulanase, EC 3.2.1.41), which is prominent in the starch processing industry because it can attacks both α‐1,6 linkages in pullulan and α‐1,4 linkages of other branched substrates to produce glucose, maltose and maltotriose; (iii) pullulan hydrolase type I (neopullulanase, EC 3.2.1.135), cleaving only α‐1,4 linkages in pullulan to produce panose; (iv) pullulan hydrolase type II (isopullulanase, EC 3.2.1.57), same as pullulan hydrolase type I, only hydrolyzing α‐1,4 linkages in pullulan to produce isopanose, both enzymes sometimes referred to as cyclodextrinases (EC 3.2.1.54), because they degrade cyclodextrin faster than starch; finally, group (v) pullulan hydrolase type III, unlike the above four groups, which can attacks α‐1,4 as well as α‐1,6 glycosidic linkages in pullulan to form a mixture of maltotriose, panose, maltose and glucose . As a true pullulanases, type I is able to degrade pullulan by hydrolyzing α‐(1,6)‐glucosidic linkages of pullulan or branched substrates.…”

A novel cold-adapted type I pullulanase of Paenibacillus polymyxa Nws-pp2: in vivo functional expression and biochemical characterization of glucans hydrolyzates analysis

“…In a previous study, a pullulanase, Pul-SH3, (about 70 kDa) purified from the optimized culture of a wild strain Exiguobacterium sp. SH3 showed high catalytic activity at ambient temperature and seemed to exhibit outstanding cold-adapted characteristics [ 8 ]. However, the gene of the enzyme was not obtained and the enzymatic properties were not characterized further.…”

“…A more comprehensive knowledge on the structure and function of the cold-adapted amylopullulanase from Exiguobacterium sp. SH3 set the stage for development of customdesigned biocatalysts that are efficient at ambient temperatures [ 8 ]. Addition of a novel cold-adapted glycogen branching enzyme from Rhizomucor miehei to wheat bread increased its specific volume and had an antistaling effect in comparison with the control [ 25 ].…”

“…Also, pullulanase was used in the fermentation industry to produce low carbohydrate “light beer” by adding pullulanase with fungal α -amylase or glucoamylase to the wheat during fermentation and bioethanol production [ 6 ]. Based on the substrate specificity and end product, pullulanase have been classified into five groups including (i) pullulanase type I (EC 3.2.1.41), also called the true pullulanases, specifically hydrolyses α-1,6 glycosidic linkages in pullulan or branched oligosaccharides to produce maltotriose and linear oligosaccharides (Additional file 1 : Figure S1); (ii) pullulanase type II (amylopullulanase), attacks α-1,6 linkages in pullulan to produce maltotriose and α-1,4 linkages of other branched substrates to produce a mixture of glucose, maltose, and maltotriose (Additional file 1 : Figure S1); (iii) pullulan hydrolase type I (neopullulanase, EC 3.2.1.135), hydrolyzing α-1,4 linkages in pullulan to produce panose (Glucose-α-1,6-Glucose-α-1,4-Glucose); (iv) pullulan hydrolase type II (isopullulanase, EC 3.2.1.57), hydrolyzing α-1,4 linkages in pullulan to produce isopanose (Glucose-α-1,4-Glucose- α-1,6-Glucose); and (v) pullulan hydrolase type III, attacks α-1,4 as well as α-1,6 glycosidic linkages in pullulan forming a mixture of maltotriose, panose and maltose [ 6 – 8 ].…”

“…Most of type I pullulanase investigated exhibit thermophilic or mesophilic properties. There are just few reports of cold-adapted pullulanases, which have optimum activity at moderate temperatures and exhibit rather high catalytic activity at cold [ 8 ]. Cold-adapted enzymes are promising candidates for versatile biotechnological applications especially food industry due to the reduced risk of microbial contamination, minimized energy consumption and the fact that reacting compounds are often instable at increasing temperatures [ 17 ].…”