Production of long-chain isomaltooligosaccharides from maltotriose using the thermostable amylomaltase and transglucosidase enzymes

Amylomaltase and transglucosidase were combined to produce long-chain isomaltooligosaccharides (IMOs). IMOs are effective prebiotics that stimulate the growth of healthy bacteria in human intestines and thus promote better overall health. In this study, the p17bAMY amylomaltase was expressed from its gene, which had been directly isolated from soil samples, while transglucosidase was purchased and purified by a gel-filtration column. Crude amylomaltase was purified by heat treatment, Q-, and phenyl-sepharose column. The purified amylomaltase had a molecular weight of 57 kDa. Specificity on the substrates of the amylomaltase was also studied and it was found that this enzyme was able to catalyze transglucosylation activity using substrates G₂ to G₇. However, G₃ was the most preferred substrate for the enzyme. Here, K _m-G3 and k _cat/K _m were 23 mM and 1.72 × 10⁸ mM/min, respectively. Amylomaltase and transglucosidase were tested both alone and in combination on a G₃ substrate to study the efficient process for the IMOs production. The obtained products from the enzymatic reactions were monitored using the TLC analytical method and a densitometer. The amylomaltase led to products containing linear maltooligosaccharides, while the transglucosidase produced short-chain IMOs. Interestingly, when amylomaltase and transglucosidase were used in combination, long-chain IMOs with sizes larger than IMO₄ were observed under the determined condition.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Identification of essential histidines in cyclodextrin glycosyltransferase isoform 1 from Paenibacillus sp. A11

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-beta-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants (k(inactivation)) of 29.5 M(-1)s(-1). The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of 3,200 M(-1)cm(-1). It was discovered that methyl-beta-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with R(t) 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.     

Insights into antifolate resistance from malarial DHFR-TS structures

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. The efficacy of this class of DHFR-inhibitor drugs is now compromised because of mutations that prevent drug binding yet retain enzyme activity. The crystal structures of PfDHFR-TS from the wild type (TM4/8.2) and the quadruple drug-resistant mutant (V1/S) strains, in complex with a potent inhibitor WR99210, as well as the resistant double mutant (K1 CB1) with the antimalarial pyrimethamine, reveal features for overcoming resistance. In contrast to pyrimethamine, the flexible side chain of WR99210 can adopt a conformation that fits well in the active site, thereby contributing to binding. The single-chain bifunctional PfDHFR-TS has a helical insert between the DHFR and TS domains that is involved in dimerization and domain organization. Moreover, positively charged grooves on the surface of the dimer suggest a function in channeling of substrate from TS to DHFR active sites. These features provide possible approaches for the design of new drugs to overcome antifolate resistance.     

The role of tryptophan-48 in catalysis and binding of inhibitors of Plasmodium falciparum dihydrofolate reductase

Dihydrofolate reductases (DHFRs) from Plasmodium falciparum (Pf) and various species of both prokaryotic and eukaryotic organisms have a conserved tryptophan (Trp) at position 48 in the active site. The role in catalysis and binding of inhibitors of the conserved Trp48 of PfDHFR has been analysed by site-specific mutagenesis, enzyme kinetics and use of a bacterial surrogate system. All 19 mutant enzymes showed undetectable or very low specific activities, with the highest value of k(cat)/K(m) from the Tyr48 (W48Y) mutant (0.12 versus 11.94M(-1)s(-1)), of about 1% of the wild-type enzyme. The inhibition constants for pyrimethamine, cycloguanil and WR99210 of the W48Y mutants are 2.5-5.3 times those of the wild-type enzyme. All mutants, except W48Y, failed to support the growth of Escherichia coli transformed with the parasite gene in the presence of trimethoprim, indicating the loss of functional activity of the parasite enzyme. Hence, Trp48 plays a crucial role in catalysis and inhibitor binding of PfDHFR. Interestingly, W48Y with an additional mutation at Asn188Tyr (N188Y) was found to promote bacterial growth and yielded a higher amount of purified enzyme. However, the kinetic parameters of the purified W48Y+N188Y enzyme were comparable with W48Y and the binding affinities for DHFR inhibitors were also similar to the wild-type enzyme. Due to its conserved nature, Trp48 of PfDHFR is a potential site for interaction with antimalarial inhibitors which would not be compromised by its mutations.     

Expression of cyclodextrinase gene from Paenibacillus sp. A11 in Escherichia coli and characterization of the purified cyclodextrinase

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and 40 degrees C. The catalytic efficiency (k(cat)/K(m)) values for alpha-, beta-, and gamma- CD were 3.0 x 10(5), 8.8 x 10(5), and 5.5 x 10(5) M(-1) min(-1), respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.     

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC₅₀ in the 28-695 μM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.     

Enzymatic synthesis of propyl-α-glycosides and their application as emulsifying and antibacterial agents

Alkyl glycosides have been effectively used in many industries because of their biodegradable, emulsification and antibacterial properties. In this study, the alkyl glycoside of propyl glycosides (PG_n) was synthesized using β-cyclodextrin (β-CD) and 1-propanol through the transglycosylation reaction of recombinant cyclodextrin glycosyltransferase (CGTase) from the Bacillus circulans A11. The optimal condition for the synthesis of propyl glycosides consisted of an incubation of 1.5% (w/v) β-CD and 500 U/mL of CGTase in a water/propanol content containing 10% (v/v) 1-propanol at pH 6.0, 50°C for 96 h. Upon analysis of the product at the optimal condition by TLC, at least three products which move faster than glucose were observed. These transferred products were formed with molecular weights of 222.1, 384.1 and 546.4 daltons as determined by mass spectrometry analysis; these values were in accordance with propyl glucoside (PG₁), propyl maltoside (PG₂) and propyl maltotrioside (PG₃), respectively. PG₁ and PG₂ were produced and prepared on a large scale and subsequently purified by preparative TLC. The combined ¹H- and ¹³C-NMR analysis confirmed that the structures of PG₁ and PG₂ were propyl-α-D-glucopyranoside and propyl-α-D-maltopyranoside, respectively. Both PG₁ and PG₂ showed emulsification activity and stability in their formation in water and n-hexadecane. Furthermore, the antibacterial activity of both products was determined and it was found that PG₂ had a higher antibacterial activity against Staphylococcus aureus and Escherichia coli than that of PG₁.     

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

Influenza neuraminidase (NA) proteins expressed in TK⁻ cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.     

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k_cat/K_m of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.