No abstract

To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various β-mannanases, a piece of loop-structure with seven amino acids between two β-strands (βD and βE) in the substrate-binding groove, named "Loop DE," was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 β-mannanases. Therefore, three AuMan5A's mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence ((316)KSPDGGN(322)) of AuMan5A with the corresponding sequences of other three family 5 β-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T opt) of 75 °C, T m value of 76.6 °C and half-life (t 1/2) of 480 min at 70 °C, which were 10 and 12.1 °C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k cat/K m) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T opt and t 1/2 of expressed re-AuMan5A-Af(D320G) decreased to 70 °C and 40 min, respectively, while its k cat/K m was only 35 % of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.

New alpha-thiohemiaminals, 7alpha-phenylthio-7-epideoxynupharidin-6-ol, the diasteriomeric 7beta-phenylthiodeoxy-nupharidin-6-ol, and 3-methyl-3-methylthio-4-hydroxyquinolizidine, were prepared and reduced to the corresponding beta-tert-amino sulfides. The configuration at C-7 of the beta-tert-amino sulfides was determined by observing the direction of the solvent-induced shift of the C-7 methyl proton resonance. The configuration at C-7 of the new alpha-thiohemiaminals was established by correlations with the beta-tert-amino sulfides and confirmed, in the case of the deoxynupharidine derivatives, by circular dichroism and ascertaining the sterochemistry of deuteride incorporation upon sodium borodeuteride reduction of the alpha-thiohemiaminal. The in vitro antifungal activities of six compounds possessing the quinolizidine skeleton, including all the newly synthesized compounds as well as some previously reported ones, and amphotericin B were tested against several human pathogenic fungi. Besides amphotericin B, only the two deoxynupharidine alpha-thiohemiaminals were active, especially against Histoplasma capsulatum and Blastomyces dermatitidis. The observations indicate activity is derived from the 3-furyl group and the functionality from which alpha-thioimmonium ions can be produced.