Yuxiang Bai

D-Allulose 3-epimerase (DAEase) catalyzes d-fructose conversion to d-allulose, but the poor thermostability of Clostridium cellulolyticum H10 DAEase limits its industrial application. Here, we enhanced DAEase thermostability by targeting the subunit interface using PROSS-guided combinatorial engineering and spatial clustering. Candidate mutations were classified into interface core, interface-adjacent, and distal regions, followed by stepwise iterative combination. Two mutants, M5 and M6, retained WT-like activity but showed markedly improved thermostability. The Tm values of M5 and M6 increased by 11.4 and 12.4 °C, respectively, while their half-lives at 65 °C increased 3-fold and 12-fold. Structural analysis indicated that interface mutations promoted salt-bridge reconstruction, distal mutations stabilized monomers, and interface-adjacent mutations optimized the assembly microenvironment. This spatially coordinated strategy provides an effective approach for engineering thermostable multimeric enzymes.

β-Agarase plays a crucial role in preparing bioactive agar oligosaccharides, but its insufficient thermostability limits industrial application. In this study, we developed a stepwise design strategy integrating multitool consensus prediction, structure-based rational screening, and greedy algorithm-based optimization to enhance AgaDcat's thermostability. This yielded mutant M3 (N120S-D243N-Q246A-S287E-A335D), which exhibits an 11 °C higher melting temperature (T_m) and 14-fold longer half-life (t_1/2) at 50 °C than the wild-type. Molecular dynamics simulations showed that mutations strengthened hydrophobic interactions, salt bridges, and hydrogen bond networks, while optimizing surface charge. The M3 variant performed well in high-temperature agarose hydrolysis, mainly producing neoagarotetraose (NA4) and neoagarohexaose (NA6), showing great industrial potential. This framework improves the efficiency of enzyme thermostability engineering and provides a general approach for industrial enzymes modification.

Cyclodextrins (CDs) are cyclic oligosaccharides composed of α(1 → 4) linked glucose units, which are widely used as solubilizers and stabilizers in the food, pharmaceutical and cosmetic industries. Among the CDs, γ-CD has attracted much attention due to its larger hydrophobic cavity and higher solubility. However, the industrial production of γ-CD is limited by lack of suitable enzymes and production process shortcomings. In this study, various strategies of improving heterologous enzyme production and optimization of the starch conversion process were applied to increase the production of γ-CD. A γ-cyclodextrin glucanotransferase with good product specificity from Bacillus sp. FJAT-44876 (BFγ-CGTase) and a liquefying β-CGTase from Bacillus sp. 1011 (Bsβ-CGTase) were successfully secreted by Pichia pastoris. After codon optimization and using the one-factor-at-a-time (OFAT) principle to improve the fermentation, the yield of recombinant BFγ-CGTase was increased 13.3 times to 463 U/L. Next a process was established involving Bsβ-CGTase-assisted starch liquefaction and simultaneous pullulanase debranching and BFγ-CGTase production of γ-CD. The yield of γ-CD increased by 17.67% via optimizing the amounts of BFγ-CGTase and BtPul used for the reaction. Overall, combination of the various improvements provided a new process for efficient preparation of γ-CD.

The GH70 family 4,3/6-α-glucanotransferase efficiently converted readily digestible (α1→4) glycosidic bonds into (α1→3) or (α1→6) glycosidic bonds, thus serving as an effective enzymatic tool for preparing starch-based dietary fiber. Amino acid residues, including V1409, Q1397, T1366, K1362, H1369, and N1418 of Limosilactobacillus fermentum NCC 2970 GtfB 4,3-α-glucanotransferase (Lf2970 GtfB), were found via sequence conservation analysis and tunnel engineering for further analysis. Mutants from K1362, T1366, H1369, and N1418 at the tunnel entrance showed increased enzyme activity and catalytic efficiency but reduced molecular weight of products. The mutant Q1397P within the tunnel increased the proportion of (α1→6) bonds in the products to 7.9% and (α1→3) bonds to 26.3%, from the wild-type of 1.2% and 21.9%, respectively. These results indicated that amino acid residues located at the tunnel entrance regulated the enzyme activity and product size of Lf2970GtfB, while the Q1397 site within the tunnel regulated the type and proportion of glycosidic bonds.

This study develops a multienzyme coimmobilization strategy on NTA-functionalized ZIF-8-coated magnetic nanoparticles (NZMNPs) for efficient d-allulose synthesis. Under optimized immobilization conditions (enzyme-to-carrier ratio: 1:50 w/w, 30 min immobilization), the system achieved an immobilization efficiency of 93.7% along with 107.1% activity retention. Immobilization markedly enhanced the enzyme stability and reusability. The immobilized d-allulose 3-epimerase (DPE) retained 60% of its initial activity after 360 min at 55 °C─more than double the residual activity of the free enzyme (26.8%)─and maintained 65.2% activity after 20 cycles of reuse. Coimmobilization of DPE, l-rhamnulose kinase (RhaB), and polyphosphate kinase (PPK) achieved a 78.4% d-allulose conversion yield and retained 53.1% residual activity after five operational cycles. The poor thermostability of RhaB affected the overall catalytic efficiency of the multienzyme cascade. To address the thermal instability of RhaB, a PROSS strategy was employed. The resulting S44K mutant significantly improved cascade stability at 30 °C. Taken together, these findings confirm that NZMNPs serve as a robust and efficient platform for multienzymes coimmobilization, highlighting their potential for industrial d-allulose production.

Reuteran is a functional homopolysaccharide comprising major (alpha 1 -> 4) and interspersed (alpha 1 -> 6) linkages whose properties and functions are directly associated with the structure. In order to enrich the structural variety and application prospects of reuteran, a GtfB-type 4,6-alpha-glucanotransferase from Limosilactobacillus mucosae L24-B (LmGtfB), which can synthesize reuteran with high-content linear (alpha 1 -> 6) linkages, was screened. LmGtfB exhibited 4,6-alpha-glucanotransferase activity against starch or dextrin and can synthesize both linear and branching (alpha 1 -> 6) linkages, which offered advantages in terms of source and cost. The as-produced reuteran had the highest content of linear (alpha 1 -> 6) linkages (19% in the methylation result) among the reuterans synthesized by other GtfB-type 4,6-alpha-glucanotransferases. A purification method was established and applied to the extraction of the core structure of the LmGtfB products. 1H NMR and enzymatic hydrolysis revealed that the core structure contains 46.24% (alpha 1 -> 6) linkages and alternating (alpha 1 -> 4)/(alpha 1 -> 6) linkages, which was formed by enzymatic transfer of maltose.