The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS-PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k cat and K m of the purified enzyme with pullulan as substrate were approximately 79 s À1 and 1.284 mg ml À1 , respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 Å . The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 Å resolution at a synchrotron and is suitable for structure determination.
Crystal structures of Klebsiella pneumoniae pullulanase (KPP) in complex with α‐cyclodextrin (α‐CD), β‐cyclodextrin (β‐CD) and γ‐cyclodextrin (γ‐CD) were refined at around 1.98–2.59 Å resolution from data collected at SPring‐8. In the structures of the complexes obtained with 1 mM α‐CD or γ‐CD, one molecule of CD was found at carbohydrate‐binding module 41 only (CBM41). In the structures of the complexes obtained with 1 mM β‐CD or with 10 mM α‐CD or γ‐CD, two molecules of CD were found at CBM41 and in the active‐site cleft, where the hydrophobic residue of Phe746 occupies the inside cavity of the CD rings. In contrast to α‐CD and γ‐CD, one β‐CD molecule was found at the active site only in the presence of 0.1 mM β‐CD. These results were coincident with the solution experiments, which showed that β‐CD inhibits this enzyme more than a thousand times more potently than α‐CD and γ‐CD. The strong inhibition of β‐CD is caused by the optimized interaction between β‐CD and the side chain of Phe746. The increased Ki values of the F746A mutant for β‐CD supported the importance of Phe746 in the strong interaction of pullulanase with β‐CD.
Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.
The research about analysis of cadmium (Cd) and lead (Pb) metal content on sediment inner part of Ambon Bay has been done. Sampling point located at Dermaga Ferry Poka, PLTD Poka, Kate-kate, Waiheru, Lateri III, Passo dan PLTD Galala. The results of analyzed of sediment particle size inside the Ambon Bay showed 3 components were sand, gravel, and sludge, but the sediments was dominated by sludge sand. Analysis of Cd content showed that at 7 stations in Ambon Bay detected Cd metal with the highest concentration at 4th station of Waiheru village was 0,6640 mg/Kg and the lowest concentration at 3th station of Kate-kate village was 0,0980 mg/Kg. Meanwhile, the highest Pb content was 20,075 mg/kg at 4th station and the lowest was 5,755 mg/kg at 2nd station of PLN Poka. The content of elements composition of sediment are SiO2, K2O, P2O5, Fe2, BaO, NiO and Ga2O3 but Cd and Pb metal was not detected.
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