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A carbohydrate binding module 68 (CBM68) of pullulanase from Anoxybacillus sp. LM18-11 was used to enhance the secretory expression of a thermostable α-amylase (BLA702) in Bacillus subtilis, through an atypical secretion pathway. The extracellular activity of BLA702 guided by CBM68 was 1248 U/mL, which was 12.6 and 7.2 times higher than that of BLA702 guided by its original signal peptide and the endogenous signal peptide LipA, respectively. A single gene knockout strain library containing 51 genes encoding macromolecular transporters was constructed to detect the effect of each transporter on the secretory expression of CBM68-BLA702. The gene knockout strain 0127 increased the extracellular amylase activity by 2.5 times. On this basis, an engineered strain B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was constructed by integrating BLA702 and CBM68-BLA702 at the AmyE and NprB sites in the genome of B. subtilis 0127, respectively. The molecular chaperone PrsA was overexpressed, to reduce the inclusion body formation of the recombinant enzymes. The highest extracellular amylase activity produced by B. subtilis 0127 (AmyE::BLA702-NprB::CBM68-BLA702-PrsA) was 3745.7 U/mL, which was a little lower than that (3825.4 U/mL) of B. subtilis 0127 (pMAC68-BLA702), but showing a better stability of passage. This newly constructed strain has potential for the industrial production of BLA702. [ABSTRACT FROM AUTHOR]

Corn starch was dually modified using thermostable α-amylase and pullulanase to prepare resistant starch (RS). The concentration of starch liquid, the amount of added thermostable α-amylase, the duration of enzymatic hydrolysis and the amount of added pullulanase were optimized using RSM to increase RS content of the treated sample. The optimum pretreatment conditions were 15% starch liquid, 3 U/g thermostable α-amylase, 35 min of enzymatic hydrolysis and 8 U/g pullulanase. The maximum RS content of 10.75% was obtained, and this value was significantly higher than that of native corn starch. The degree of polymerization (DP) of the enzyme-modified starch decreased compared with that of native starch. The scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were performed to assess structural changes in native and pretreated starch. The effect of dual enzyme pretreatment on the structure and properties of corn starch was significant. Unlike the untreated one, the pretreated corn starch showed clear pores and cracks. Significant differences in RS contents and structural characterization between starch pretreated and untreated with dual enzymes demonstrated that the dual enzyme modification of corn was effective in enhancing RS contents. [ABSTRACT FROM AUTHOR]

α-amylase which catalyzes the hydrolysis of α-1,4-glycosidic bonds in starch have frequently been cloned into various microbial workhorses to yield a higher recombinant titer. A thermostable SR74 α-amylase from Geobacillus stearothermophilus was found to have a huge potential in detergent industries due to its thermostability properties. The gene was cloned into a CTG-clade yeast Meyerozyma guilliermondii strain SO. However, the CUG ambiguity present in the strain SO has possibly altered the amino acid residues in SR74 amylase wild type (WT) encoded by CUG the codon from the leucine to serine. From the multiple sequence alignment, six mutations were found in recombinant SR74 α-amylase (rc). Their effects on SR74 α-amylase structure and function remain unknown. Herein, we predicted the structures of the SR74 amylases (WT and rc) using the template 6ag0.1.A (PDB ID: 6ag0). We sought to decipher the possible effects of CUG ambiguity in strain SO via in silico analysis. They are structurally identical, and the metal triad (CaI–CaIII) might contribute to the thermostability while CaIV was attributed to substrate specificity. Since the pairwise root mean square deviation (RMSD) between the WT and rc SR74 α-amylase was lower than the template, we suggest that the biochemical properties of rc SR74 α-amylase were better deduced from its WT, especially its thermostability. [ABSTRACT FROM AUTHOR]