Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Cheng, Ya‐Shan; Ko, Tzu‐Ping; Huang, Jian‐Wen; Wu, Tzu-Hui; Lin, Cheng-Yen; Luo, Wenjun; Li, Qian; Ma, Yanhe; Huang, Chun‐Hsiang; Wang, Andrew H.J.; Liu, Je-Ruei; Guo, Rey‐Ting

doi:10.1007/s00253-011-3791-4

Cited by 33 publications

(19 citation statements)

References 38 publications

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“…3c). The optimum temperature recorded for different bacterial endoglucanases shows broad temperature values, ranging from 25 °C in Fibrobacter succinogenes (Lyo and Forsberg 1996) to 100 °C in Thermotoga maritima (Cheng et al 2012). Findings here indicate that the Spr Cel8A endoglucanase from S. proteamaculans is a mesophilic enzyme.…”

Section: Discussionmentioning

confidence: 99%

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

et al. 2016

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Serratia proteamaculans CDBB-1961, a gut symbiont from the roundheaded pine beetle Dendroctonus adjunctus, displayed strong cellulolytic activity on agar-plates with carboxymethyl cellulose (CMC) as carbon source. Automatic genome annotation of S. proteamaculans made possible the identification of a single endoglucanase encoding gene, designated spr cel8A. The predicted protein, named Spr Cel8A shows high similarity (59–94 %) to endo-1,4-β-d-glucanases (EC 3.2.1.4) from the glycoside hydrolase family 8 (GH8). The gene spr cel8A has an ORF of 1113 bp, encoding a 371 amino acid residue protein (41.2 kDa) with a signal peptide of 23 amino acid residues. Expression of the gene spr cel8A in Escherichia coli yields a mature recombinant endoglucanase 39 kDa. Cel8A displayed optimal activity at pH 7.0 and 40 °C, with a specific activity of 0.85 U/mg. The enzyme was stable at pH from 4 to 8.5, retaining nearly 40–80 % of its original activity, and exhibited a half-life of 8 days at 40 °C. The Km and Vmax values for Spr Cel8A were 6.87 mg/ml and 3.5 μmol/min/mg of protein, respectively, using CMC as substrate. The final principle products of Spr Cel8A-mediated hydrolysis of CMC were cellobiose, cello oligosaccharides and a small amount of glucose, suggesting that Spr Cel8A is an endo-β-1,4-glucanase manifesting exo-activity. This is the first report regarding the functional biochemical and molecular characterization of an endoglucanase from S. proteamaculans, found in the gut-associated bacteria community of Dendroctonus bark beetles. These results contribute to improved understanding of the functional role played by this bacterium as a symbiont of bark beetles.

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Section: Discussionmentioning

confidence: 99%

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

et al. 2016

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“…Moreover, PulA is thermostable that is beneficial for industrial usages. Solving substrate/product complex structures of industrial enzymes helps us to engineer these enzymes with better properties (e.g., higher enzyme specific activity and thermostability) . Here, the structures of apo‐PulA and its complexes with maltotriose and maltotetraose were solved at high resolution.…”

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 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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“…However, this technique requires specific protein structural information. In our previous studies, this method has proved feasible in increasing enzyme activities . On the other hand, searches for novel thermophilic enzymes from extremophiles as a starting point have been an important approach to obtain industrial useful enzymes.…”

Section: Introductionmentioning

confidence: 99%

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

et al. 2013

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Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild-type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo-form wild-type TsXylA and the xylobiose-, xylotriose-, and xylotetraose-bound E146A and E251A mutants were solved by X-ray diffraction to high resolution (1.32-1.66 Å). The protein forms a classic (β/α)8 folding of typical GH10 xylanases. The ligands in substrate-binding groove as well as the interactions between sugars and active-site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering.

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Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Cited by 33 publications

References 38 publications

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae)

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

Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL‐YS485

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