Recombinant bacterial amylopullulanases

Mohanan, Nisha; Satyanarayana, T.

doi:10.4161/bioe.24629

Cited by 56 publications

(25 citation statements)

References 81 publications

(129 reference statements)

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“…Type II pullulanases, also known as amylopullulanases, can hydrolyze both α‐1,6‐glycosidic linkages and α‐1,4‐glycosidic linkages in branched and linear oligosaccharides . All of the type I pullulanases found belong to GH13, while the amylopullulanases are classified in GH13 and GH57 based on the architecture of catalytic domain and number of conserved sequences . The GH13 family enzymes adopt a typical (α/β) 8 ‐barrel fold with the β4‐aspartate, β5‐glutamate and β7‐aspartate as the catalytic sites, and possess four to seven conserved sequences .…”

Section: Introductionmentioning

confidence: 99%

“…The GH13 family enzymes adopt a typical (α/β) 8 ‐barrel fold with the β4‐aspartate, β5‐glutamate and β7‐aspartate as the catalytic sites, and possess four to seven conserved sequences . Six amylopullulanases were classified in GH57, which adopts a (α/β) 7 ‐barrel fold with the catalytic site consisting of the strand β4‐glutamate and β7‐aspartate, and contains five conserved sequences …”

Section: Introductionmentioning

confidence: 99%

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Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11

Ren

Huang

et al. 2014

Proteins

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Pullulanase is a debranching enzyme that specifically hydrolyzes the α-1,6 glycosidic linkage of α-glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18-11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg(-1) and 16.4 μmol L(-1), respectively. PulA has a half-life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75-2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β-strand and the third α-helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate-binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18-11

Ren

Huang

et al. 2014

Proteins

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“…Pullulanase type ⌱ specifically hydrolyzes ␣-1,6 glycosidic linkages in pullulan and yields maltotriose as an end product (8). However, pullulanase type II (amylopullulanase) has an additional ability to hydrolyze ␣-1,4 glycosidic linkages in starch and other polysaccharides (9). Pullulan hydrolases (type I and type II) can only hydrolyze ␣-1,4 linkages in pullulan and are unable to hydrolyze ␣-1,6 linkages of this glucan.…”

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confidence: 99%

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

Ahmad

Rashid

Haider

et al. 2014

Appl Environ Microbiol

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cA novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and ␣-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The K m value toward pullulan was 2 mg ml ؊1 , with a V max of 109 U mg ؊1 . Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both ␣-1,6 and ␣-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was ␥-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (␣, ␤, and ␥), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.

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“…The amylopullulanases have a single active site for hydrolyzing both ␣-1,4-and ␣-1,6-glycosidic bonds, while those with two active sites for hydrolyzing ␣-1,4-and ␣-1,6-glycosidic bonds are called ␣-amylase-pullulanases [2,3]. The amylopullulanases find applications in the starch industry for one-step starch liquefaction-saccharification for making various sugar syrups, and as antistaling agent in bread and detergent additive [4].…”

Section: Introductionmentioning

confidence: 99%