Caiming Li

Calcium ion contribution to thermostability of cyclodextrin glycosyltransferase is closely related to calcium-binding site CaIII.

In the study, we investigated the contribution of Ca²⁺ to the thermostability of α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans , which has two calcium-binding sites (CaI and CaII), and β-CGTase from Bacillus circulans , which contains an additional calcium-binding site (CaIII), consisting of Ala315 and Asp577. It was found that the contribution of Ca²⁺ to the thermostability of two CGTases displayed a marked difference. Ca²⁺ affected β-CGTase thermostability significantly. After Ca²⁺ was added to β-CGTase solution to a final concentration of 5 mM followed by incubation for 120 min at 60 °C, residual activity of β-CGTase was 88.3%, which was much higher than that without Ca²⁺. However, Ca²⁺ had a small contribution to α-CGTase thermostability. Furthermore, A315D and D577K mutations at CaIII could significantly change the contribution of Ca²⁺ to β-CGTase thermostability. These results suggested that the contribution of Ca²⁺ to CGTase thermostability was closely related to CaIII.

Effects of montmorillonite addition on the performance of starch-based wood adhesive

Effects of montmorillonite (MMT) addition on the performance of corn starch-based wood adhesive were investigated. It was found that MMT addition could enhance the shear strength of the starch-based wood adhesive. The shear strength of the adhesive with 5% (w/w, dry starch basis) MMT reached 10.6 MPa in the dry state, which was almost twice that of the same adhesive without MMT. Addition of 5% MMT also produced an approximately 1.2-fold increase in the shear strength in the wet state. Although this addition caused an increase in the viscosity, the resulting adhesive retained both good mobility and viscosity stability during storage. MMT also enhanced the shear-thinning and solid-like behaviors of the adhesive, compared with the adhesive without MMT. Finally, MMT addition improved the thermal stability of the adhesive. In conclusion, addition of MMT to starch-based wood adhesives can improve their overall performance, enhancing their value as alternatives for traditional petrochemical-based wood adhesives.

Mutations enhance β-cyclodextrin specificity of cyclodextrin glycosyltransferase from Bacillus circulans

The effects of amino acid residue at position 31 in the neighborhood of calcium binding site I (CaI) on product specificity of cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) were investigated by replacing Ala31 in the CGTase from Bacillus circulans STB01 with arginine, proline, threonine, serine and glycine. The results showed that the mutations A31R, A31P, and A31T resulted in the increases in β-cyclodextrin-forming activity and β-cyclodextrin production, indicating that these mutations enhanced β-cyclodextrin specificity of the CGTase. Especially the mutant A31R displayed approximately 26% increase in β-cyclodextrin production with a concomitant 41% decrease in α-cyclodextrin production when compared to the wild-type CGTase. Thus, it was much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhanced β-cyclodextrin specificity of the mutants might be a result of stabilizing CaI, which also suggested that CaI might play an important role in cyclodextrin product specificity of CGTase.

Mutations in cyclodextrin glycosyltransferase from Bacillus circulans enhance β-cyclization activity and β-cyclodextrin production.

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is used to produce cyclodextrins, which are cyclic glucans with many industrial applications. In the present study, the effects of the amino acid residue at position 577, which is located in calcium-binding site III (CaIII), on cyclization activity and cyclodextrin production were investigated by replacing Asp577 in CGTase from Bacillus circulans STB01 with glutamate, arginine, lysine, and histidine. The results showed that mutations D577E and D577R significantly increased the β-cyclization activity. The D577R mutant, in particular, displayed a 30.7% increase in the β-cyclization activity when compared to the wild-type CGTase. Furthermore, under conditions resembling industrial production processes, the D577R and D577E mutants displayed 9.1 and 2.0% enhancement in β-cyclodextrin production, respectively. More importantly, the higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the process. Thus, the two mutants were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme.

Polyethylene glycols enhance the thermostability of β-cyclodextrin glycosyltransferase from Bacillus circulans.

We investigated the ability of six polyethylene glycols (PEGs), with molecular weights ranging from 400 to 20,000 Da, to enhance the thermostability of β-cyclodextrin glycosyltransferase (β-CGTase) from Bacillus circulans. We found that PEGs with different molecular weights could activate and stabilize this β-CGTase, but to different degrees. The most significant increase (about 20%) in β-cyclodextrin-forming activity was achieved by adding 10-15% PEG 400. PEGs with low molecular weights also significantly enhanced the thermostability of β-CGTase; 15% PEG 1000 prolonged its half-life at 60°C by 6.5-fold, compared to a control. Fluorescence spectroscopy and circular dichroism analysis indicated that PEGs helped protect the tertiary and secondary structure of β-CGTase, respectively. This study provides an effective approach for improving the thermostability of CGTases and related enzymes.

Preparation and characterization of pullulanase debranched starches and their properties for drug controlled-release

Debranched starches (DBSs) with different degrees of debranching (low, L-DBS; moderate, M-DBS; high, H-DBS) were prepared and investigated. After pullulanase modification, the starch granules became more porous and many small particles containing short glucan chains were generated. DBSs adopt a single-helical V-type crystalline structure with low crystallinity. L-DBS samples contained fewer (20.70%) and longer (degree of polymerization, DP: 21.92) linear short glucan chains than their counterparts (M-DBS: 40.92%, 20.05 DP; H-DBS: 55.52%, 18.52 DP). Pullulanase enzymatic hydrolyzate for DBS samples with higher degrees of debranching inclined towards retrogradation at 20 °C. DBSs with higher degrees of debranching could form a hydrogel with higher G′ and G′′ values, indicating these samples formed a stronger gel network. L-DBS could hold more water and its digestibility was higher. The in vitro test showed that DBS is a good candidate to control drug release for over 12 h. Furthermore, the drug release profiles from both DBS-based and HPMC-based tablets showed an anomalous transport mechanism. The drug release from these four matrices was controlled by a combination of drug diffusion and matrix erosion. The drug release properties from DBS-based tablets were considerably influenced by the degree of debranching. The in vitro drug release profile of M-DBS was similar to that of HPMC (f2 = 60.75), while L-DBS and H-DBS differed from HPMC (f2 < 50). In summary, DBS is a good hydrogel candidate, and it can be used as an excipient in oral tablets to control drug release.

Effects of heat pretreatment of starch on graft copolymerization reaction and performance of resulting starch-based wood adhesive

In this study, effects of starch heat pretreatment at 70, 80 and 90°C on graft copolymerization reaction with vinyl acetate (VAc) and the performance of the resulting starch-based wood adhesive (SWA) were investigated. It was shown that SWA pretreated at 90°C achieved the best performance. At this temperature, the bonding capacity improved by 17.84% compared to the adhesive synthesized without heat pretreatment and the viscosity increased by 18.16% after 7 free-thaw cycles, much better than other samples. Scanning electron microscopy (SEM) and polarizing microscopy demonstrated that structures of starch granules were fully damaged after heat pretreatment at 90°C. The reaction took place not only on the surface of starch granules, but also internally, leading to improvement in the grafting amounts and grafting efficiency by 42.86% and 39.03%, respectively. This was further confirmed by Fourier transform infrared spectroscopy (FT-IR), Confocal Raman microscopy (CRM) and X-ray photoelectron spectroscopy (XPS), which also showed better reaction homogeneity both between different starch granules and from granule surface to its internal structure.

Pasting and thermal properties of waxy corn starch modified by 1,4-α-glucan branching enzyme

Waxy corn starch was modified with the 1,4-α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02. Incubating waxy corn starch with GBE increased the number of α-1,6 branch points and reduced the average chain length. Enzymatic modification also decreased the breakdown and setback values of Brabender viscosity curves, indicating that the modified starch had higher paste stability. Preheating the starch at 65°C for 30min before incubation with GBE could promote enzymatic modification of starch. Linear regression was used to describe the relationships between starch structure and its pasting and thermal properties. The setback value showed a negative linear correlation with the α-1,6 branch point content (R2=0.9824) and a positive linear correlation with the average chain length (R2=0.8954). Meanwhile, the gelatinization enthalpy was also linearly correlated to the α-1,6 branch point content (R2=0.9326) and the average chain length (R2=0.8567). These insights provide a useful reference for food processors.

Characterisation of physicochemical and functional properties of soluble dietary fibre from potato pulp obtained by enzyme-assisted extraction

In this study, enzyme-assisted methodology was applied with the aim to optimise the efficiency of soluble dietary fibre (SDF) extraction from potato pulp. The yields of SDF pretreated by cellulase, xylanase, and a cellulase/xylanase mixture were 31.9% (w/w), 25.7% (w/w), and 39.7% (w/w) respectively. When comparing single enzyme pretreatment with combined enzymatic treatment, we observed differences in properties related to molecular weight and viscosity. SDF obtained by xylanase pretreatment rendered better results for glucose dialysis retardation index (32.98%, w/w), amylase inhibitory effect (56.2%), and pancreatic lipase inhibitory activity (55.33%), while combined enzymatic pretreatment exhibited better effect in properties related to exposure of functional groups and specific surface area, including sodium cholate binding capacity (72.2%) and hydroxyl radical scavenging activity (87.57%). Our results indicated that enzymatic pretreatment, which could effectively hydrolyse cellulose and hemicellulose components, could not only benefit the yield of SDF, but also enhance its physiological and functional properties.

Binary and Tertiary Complex Based on Short-Chain Glucan and Proanthocyanidins for Oral Insulin Delivery

The present study was performed to investigate binary and tertiary nanocomposites between short-chain glucan (SCG) and proanthocyanidins (PAC) for the oral delivery of insulin. There was a large decrease in fluorescence intensity of insulin in the presence of SCG or the combination of SCG with PAC. Fourier transform infrared spectroscopy revealed that the binary and tertiary nanocomposites were synthesized due to the hydrogen bonding and hydrophobic interactions. The insulin entrapped in the nanocomposites was in an amorphous state confirmed by X-ray diffraction. The cell culture demonstrated that both the nanocomposites showed no detectable cytotoxicity with relative cell viability all above 85%. The pharmacological bioavailability after oral administration of insulin-SCG-PAC at a dose of 100 IU/kg was found to be 6.98 ± 1.20% in diabetic rats without any sharp fluctuations in 8 h.