D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K_m and k_cat/K_m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.
Recombinant Proteins/metabolism* ; Paenibacillus/metabolism* ; Mannose/metabolism* ; Escherichia coli/metabolism* ; Mannitol/metabolism*

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.
Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Paenibacillus ; enzymology ; Recombinant Proteins ; genetics ; gamma-Cyclodextrins ; metabolism

Auxotrophic strains of N1-37 (Phe-) and N2-27 (His-), screened from mutations of Paenibacillus polymyxa JSa-9 previously, were used as the parent strains to screen high-producing LI-F antibacterial lipopeptide fusion strain through protoplast fusion with polyethylene glycol as a promote agent. Fusion strain F5-15 was obtained. Then the product of LI-F antibacterial lipopeptide was quantified by HPLC, and the difference of expression of the key genes of lipopeptide synthase between wild strain JSa-9 and the fusion strain was analyzed by real-time PCR. LI-F antibacterial lipopeptide yield of the fusion strain F5-15 was 3.1-fold of the original strain JSa9's, and the expression levels of the target genes were 10.48, 2.48, 2.1 and 11.8 fold of the initial strain JSa-9, respectively.
Anti-Bacterial Agents ; biosynthesis ; Chromatography, High Pressure Liquid ; Lipopeptides ; biosynthesis ; Paenibacillus ; metabolism ; Protoplasts ; metabolism ; Real-Time Polymerase Chain Reaction

With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
Antifibrinolytic Agents ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fibrinolytic Agents ; metabolism ; Genetic Vectors ; genetics ; Paenibacillus ; chemistry ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

By engineering the subsite +1 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans, we improved its maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) synthesis. Specifically, we conducted site-saturation mutagenesis on Leu194, Ala230, and His233 in subsite +1 separately and gained 3 mutants L194N (leucine --> asparagine), A230D (alanine --> aspartic acid), and H233E (histidine --> glutamic acid) produced higher AA-2G yield than the wild-type and the other mutant CGTases. Therefore, the 3 mutants L194N, A230D, and H233E were further used to construct the double and triple mutations. Among the 7 obtained combinational mutants, the triple mutant L194N/A230D/H233E produced the highest AA-2G titer of 1.95 g/L, which was increased by 62.5% compared with that produced by the wild-type CGTase. Then, we modeled the reaction kinetics of all the mutants and found a substrate inhibition by high titer of L-AA for the mutants. The optimal temperature, pH, and reaction time of all the mutants were also determined. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the three positions (194, 230 and 233) and the substrate sugars.
Ascorbic Acid ; analogs & derivatives ; chemistry ; Glucosyltransferases ; genetics ; metabolism ; Hydrogen Bonding ; Kinetics ; Mutagenesis, Site-Directed ; Paenibacillus ; enzymology ; Polysaccharides ; chemistry ; Protein Engineering ; Substrate Specificity ; Temperature

To enhance the thermostability and storage stability of alpha-cyclodextrin glycosyltransferase (a-CGTase), we added specific chemical additives into the preparation of alpha-CGTase, and studied the effect of additives on the storage stability of alpha-CGTase at different temperatures. Then we measured the protein structure of CGTase in the far UV (200-250 nm) and near UV (250-320 nm) ranges respectively by Circular dichroism (CD) spectra under high temperature and analyzed the relationship between thermostability and protein structure. The results indicated that the addition of selected additives (gelatin, glycerin, CaCl2 and PEG400) enhanced the thermostability of alpha-CGTase dramatically. After 45 days, the preparation of alpha-CGTase still had 100% of the enzyme activity with different additives superimposed at the optimum concentration at 40 degrees C. The CD spectra of alpha-CGTase showed that glycerin could protect the secondary and the tertiary structure of the CGTase under high temperature and therefore the enzyme maintained its high activity. Chemical additives can improve the stability of alpha-CGTase significantly and they preserve the enzyme activity by protecting its secondary structure.
Enzyme Stability ; drug effects ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; chemistry ; genetics ; Glycerol ; chemistry ; Paenibacillus ; enzymology ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

The cgt gene was isolated from Paenibacillus macerans by PCR amplification and was inserted into vectors of pPIC9K and pMAS. The recombinant vectors were transformed to Pichia pastoris KM71 and Bacillus subtilis WB600, respectively. The results showed that alpha-CGTase activity in the culture media of recombinant P pastoris was only 0.2 U/mL, while it was 1.9 U/mL in recombinant B. subtilis. In addition, we optimized the culture conditions of the recombinant B. subtilis strain. After cultivation at 37 degrees C for 24 h with shake flask, the CGTase forming activity in culture media reached to 4.5 U/mL (hydrolysis activity was 3200 IU/mL), which is 9.8-fold to that of the original strain P. macerans.
Amino Acid Sequence ; Bacillus subtilis ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; genetics ; metabolism ; Molecular Sequence Data ; Paenibacillus ; enzymology ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics