A novel thermostable type I pullulanase gene ( pul ) from Geobacillus thermocatenulatus DSMZ730 was cloned. It has an open reading frame of 2154 bp encoding 718 amino acids. G. thermocatenulatus pullulanase (Pul) was found to be optimally active at pH 6.5 and 70 °C. It exhibited stable activity in the pH range of 5.5-7.0. Pul lacked three domains (CBM41 domain, X25 domain, and X45 domain) compared with the pullulanase from Bacillus acidopullulyticus ( 2WAN ). Different N-terminally domain truncated (730T) or spliced (730T-U1 and 730T-U2) mutants were constructed. Truncating the N-terminal 85 amino acids decreased the K value and did not change its optimum pH, an advantageous biochemical property in some applications. Compared with 2WAN , Pul can be used directly for maize starch saccharification without adjusting the pH, which reduces cost and improves efficiency.

The pullulanase gene (pulGK), encoding a thermostable type I pullulanase (PulGK), is obtained from the strain Geobacillus kaustophilus DSM7263. The gene has an open reading frame of 2157 bp that encodes a 718‐amino‐acid pullulanase, and shows the highest identity with the pullulanase from Geobacillus thermoleovorans US105. The pulGK is expressed in Bacillus subtilis WB800N using the plasmid pHT43, and the recombinant protein is secreted using the amyQ signal peptide. The level of PulGK produced in B. subtilis reaches 0.08 mg mL−1 after induction for 40 h at 30 °C. The purified recombinant PulGK can attack the α‐1,6 linkages specifically in pullulan to generate maltotriose as the major product. Its specific activity is observed to be 64.75 U mg−1 and the Km and Vmax values of purified PulGK are 11.7 mg mL−1 and 23.6 μmol min−1. Purified PulGK shows optimal activity at pH 6.0 and 65 °C. It also shows significant thermostability, with a T1/2 of 60 h at 65 °C. Recombinant PulGK is immobilized and the thermostability of immobilized PulGK (Im‐PulGK) is significantly improved (55–75 °C). PulGK hydrolyzes pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and product analysis proves that the purified pullulanase from Geobacillus kaustophilus DSM7263 belongs to a type I pullulanase. This is the first report of pullulanase from Geobacillus kaustophilus (which includes the wild strain and the recombinant production of the enzyme) with detailed enzymatic properties of heterologous expression. The significant thermostability and production of recombinant pullulanase by B. subtilis may also potentially prove to be valuable in industrial applications.

This work presents a synthetic route to produce chloramphenicol esters by taking advantage the high enantio- and regio-selectivity of lipases. A series of chloramphenicol esters were synthesized using chloramphenicol, acyl donors of different carbon chain length and lipase LipBA (lipase cloned from Bacillus amyloliquefaciens). Among acyl donors with different carbon chain lengths, vinyl propionate was found to be the best. The influences of different organic solvents, reaction temperature, reaction time, enzyme loading and water content on the synthesis of the chloramphenicol esters were studied. The synthesis of chloramphenicol propionate (0.25 M) with 4.0 g L−1 of LipBA loading gave a conversion of ~98% and a purity of ~99% within 8 h at 50 °C in 1,4-dioxane as solvent. The optimum mole ratio of vinyl propionate to chloramphenicol was increased to 5:1. This is the first report of B. amyloliquefaciens lipase being used in chloramphenicol ester synthesis and a detailed study of the synthesis of chloramphenicol propionate using this reaction. The high enzyme activity and selectivity make lipase LipBA an attractive catalyst for green chemical synthesis of molecules with complex structures.

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Supplementary Figure from EXOC4 Promotes Diffuse-Type Gastric Cancer Metastasis via Activating FAK Signal