Molecular Catalysis
·
2024
·
IF 3.9
Wenhao Xia,Shimeng Liu,Lihui Huang,Hao Wu,Tao Bai,Shanshan Wang,Fei Hao,Xiao Wang,Wen Wang
d -tagatose, a rare sugar with broad applications in the food and pharmaceutical industries, can be synthesized from d -fructose by epimerases, an approach with significant biosynthesis potential. However, so far there has been only one attempt to engineer tagatose 4‑epimerase through random mutagenesis. The lack of exploration has severely restricted the application of epimerases in the industrial production of d -tagatose. The previous research primarily focused on enhancing the catalytic activity of epimerases through random mutagenesis, with limited understanding of how substrate-adjacent residues affect enzyme efficiency, which was hence the subject of this study. First, using a semi-rational design approach, after three rounds of mutagenesis and screening, the mutant (S125D/S268A/Q124E) of D-tagaturonate epimerase (UxaE) from Thermotoga neapolitana was obtained, which increased d -tagatose production efficiency by 10.8-fold compared to the wild type, with a specific activity of 1505.6 U/g, representing the highest specific activity reported for a UxaE mutant to date. Molecular dynamics simulations showed that the substitution of Ser to Asp-125 enhanced substrate-enzyme interactions, while the substitution of Glu to Gln-124 reinforced the hydrogen bond network around the catalytic residue Glu-128, further reshaping the substrate-binding pocket and enhancing catalytic activity. This study for the first time explored the enhancement of the C4 epimerization function of epimerases by reshaping their substrate-binding pockets, thereby providing a feasible route for the industrial biosynthesis of d -tagatose.
研究领域:
生物化学
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生物技术
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酶工程
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分子生物学
✓ 已验证
产品关联
DOI
高影响因子
Foods
·
2025
·Vol. 14
·期 11
·1873
·
IF 5.1
Zhou Chen,Yuhan Yan,Ziang Wu,Yanyin Song,Jiangqi Xu
As global milk production continues to rise, the disposal of expired milk contributes to environmental pollution and valuable resource wastage. This study presents the development of a novel L-arabinose isomerase, designatedBmAIase12, and its application in the enzymatic recycling of expired milk.BmAIase12 exhibited a specific activity of 10.7 U/mg and showed optimal performance at 50 °C and pH 7.0. Furthermore, it exhibited higher activity than most other L-arabinose isomerases. It converted D-galactose into D-tagatose with a high conversion ratio of 53.3% after 48 h at 50 °C. The conversion efficiency of expired milk to D-tagatose was recorded at 40.62%, resulting in a maximum tagatose yield of 1.625 g/L. This was accomplished through the incorporation of β-galactosidase (120 U/mL) andSaccharomyces cerevisiae(30 mg/mL) to hydrolyze lactose and metabolize glucose, followed by the addition of 3 U/mL ofBmAIase12. Ultimately, following purification, the purity of tagatose was determined to be 98%, and the final yield was 29.8%. These results suggest thatBmAIase12 may serve as a promising enzyme for D-tagatose production due to its high conversion yield.
研究领域:
生物化学
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环境科学
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食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Foods
·
2025
·Vol. 14
·期 9
·1538
·
IF 5.1
Zepeng Li,Runmin Wang,Xiantai Lai,Wenyi Liao,Runfeng Liao,Zhuohong Wu,Guoyan Zhang,Xianghui Qi
D-tagatose is a functional sweetener with glucose-regulating and prebiotic properties, but its bioproduction from D-galactose faces many limitations, particularly the high production costs. In particular, the current biosynthesis of D-tagatose suffers from thermal instability and the substrate selectivity issues of L-arabinose isomerase (L-AI) required to convert D-galactose into D-tagatose. In this study, recombinantEscherichia coliBW25113/pQE-80L-araAF118M/F279Iexpressing double mutant L-AI was immobilized to enhance its stability and reusability. The optimal conditions for whole-cell catalysis were 60 °C, pH 6.5, 5 mM Mn2+, and 20 h, with a yield of 55.2 g/L of D-tagatose. Immobilization with 3% sodium alginate and 2% CaCl2retained 90% of the production efficiency displayed by free cells. Notably, the immobilized cells exhibited enhanced heat resistance (60–70 °C) and operational stability, retaining 76% activity after five cycles. The D-tagatose production was further increased to 129.43 g/L by increasing the substrate concentration to 250 g/L. Compared to free cells, immobilized cells retained 83.6% of the initial yield up to 10 batches. This study presents a cost-effective and sustainable method for the production of D-tagatose using optimized whole-cell catalysis through immobilization, which paves the way to solve industrial challenges such as thermal instability and low substrate efficiency.
研究领域:
生物化学
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食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Foods
·
2024
·Vol. 13
·期 11
·1727
·
IF 4.7
Chengyu Lu,Ziwei Chen,Yuvaraj Ravikumar,Guoyan Zhang,Xinrui Tang,Yufei Zhang,Mei Zhao,Wenjing Sun,Xianghui Qi
L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification ofLactobacillus plantarumCY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.
研究领域:
生物化学
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食品科学
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Yuxin Wang,Zijian Tan,Hongli Wei,Nan Zhang,Lifei Wang,Yifan Yang,Weidong Liu,Leilei Zhu
D-Tagatose, a promising sugar substitute with various functional properties and commercial applications, can be enzymatically converted from D-fructose by tagatose 4-epimerase. The development of an efficient tagatose 4-epimerase that catalyzes the conversion of D-fructose into D-tagatose is essential to make the production technology of D-tagatose applicable. In this study, tagatose 4-epimerase from Thermotogae (TsT4Ease) was engineered through semi-rational design and directed evolution, resulting in a 2.8-fold improvement in catalytic activity compared to the wild type (WT). The production of D-tagatose reached 42 g/L in 2 h. Crystal structure analysis determined the structural features with a common (α/β)8-TIM barrel and a Zn2+-binding architecture at the active center. Subsequent molecular dynamics (MD) simulations revealed that the substitutions improved substrate binding energy and stabilized the active pocket. This study offers new insights into the structure–function relationship of TsT4Ease and provides a candidate tagatose 4-epimerase.
研究领域:
生物化学
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蛋白质工程
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结构生物学
✓ 已验证
DOI
PubMed
高影响因子
PLoS One
·
2025
·Vol. 20
·期 1
·e0317825
·
IF 2.6
Yuanqiang Lv,Jie Chu,Xiaoxiao Zhang,Xuan Li,Aijiao Yin,on behalf of The Industrial Microbiology Laboratory
Backgrounds
Abuse of feed supplement can cause oxidative stress and inflammatory responses in Gallus gallus . Synbiotics are composed of prebiotics and probiotics and it possess huge application potentials in the treatment of animal diseases.
Methods
This study examined the effect of d-tagatose on the probiotic properties of L . rhamnosus GG , L . paracasei , and S . lactis so as to screen the best synbiotic combinations. Treat Gallus gallus exhibiting oxidative stress and immune response caused by aflatoxin b1 with optimal synbiotics for 14 days, detect the changes of inflammatory markers and oxidative stress markers of Gallus gallus using qRT-PCR, and identified the intestinal bacteria genera and their metabolites in the cecum of Gallus gallus using gut microbiota and metabolomics analysis.
Results and conclusion
The results indicated that oxidative stress and immune response factor expressions quantity in Gallus gallus decreased significantly after 14 days of treatment, compared with model group, the low-dose treatment group’s SOD1, SOD3, GPX1, GPX2, GSR, H6DP, and HO-1 genes in liver were downregulated by 36.03%, 40.01%, 45.86%, 40.79%, 37.68%, 25.04%, and 29.89%, the IL-1, IL-2, IL-4, IL-6, IgA, IgM, and IgG genes in blood and spleen were downregulated by 26.59%, 34.19%, 21.19%, 28.18%, 35.93%, 12.67%, 21.81 and 35.93%, 22.85%, 21.19%, 28.78%, 35.93%, 15.36%, 29.73%. The intestinal bacteria genera and metabolomics analysis results indicated that the abundance of beneficial bacteria genus was up-regulated, and the proportion of pathogenic bacteria genera decreased. The amount of beneficial metabolites associated with antioxidant and anti-inflammatory effects was upregulated. The synbiotic composed of d-tagatose and L. rhamnosus GG can treat oxidative stress and immune response by altering the structure of intestinal bacteria genera and the production of metabolites.
研究领域:
动物营养学
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微生物学
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免疫学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2022
·
IF 9.231
Dongbo Wang,Ying Wang,Yuan Li,Ting Shi,Dandan Han,Junbo Gong
Impurity sugars produced in upstream process of functional sugars are significantly impacting the product quality. In this work, the effect of congeners ( d -maltose ( d -MAL), d -fructose ( d -FRU), d -glucose ( d -GLU)) on primary and secondary nucleation of d -tagatose ( d -TAG) crystals was investigated. The impurity sugars showed an inhibition on primary nucleation of d -TAG crystals, while a promotion on the secondary nucleation of d -TAG. Interestingly, the impact of impurity sugars on d -TAG crystal growth was similar to that on primary nucleation. The diffusion ability, hydrogen bonding forming ability, interaction energy of d -TAG crystal surfaces and impurity sugars were evaluated by molecular dynamics (MD) simulations to reveal the nucleation and growth behavior. Based on the above findings, we designed the d -TAG crystallization experiments, and obtained d -TAG crystals with uniform particle size distribution and regular morphology. This study helps to understand the influence of impurity sugars on crystallization, guiding the industrial manufacturing of functional sugars.
研究领域:
化学工程
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材料科学
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物理化学
✓ 已验证
DOI
PubMed
高影响因子
FOOD HYDROCOLLOIDS
·
2025
·
IF 12.4
Yan Wen,Yan Zhang,Na Lü,Zehui Xuan,Lili Lu
D-tagatose is an attractive functional sweetener, and its production via enzymatic synthesis is simple and environmentally friendly; however, it faces challenges related to enzyme and substrate costs. In this study, a novel and cost-effective method for synthesizing D-tagatose was established using L-arabinose isomerase (L-AI) immobilized within a sodium alginate (SA) composite hydrogel, with the low-cost guar gum as the starting material. Eight inorganic or organic materials were individually combined with SA to encapsulate L-AI. The immobilized enzyme prepared with SA and 50-nm SiO 2 particles demonstrated superior properties, including enhanced mechanical strength and improved reusability, compared to enzymes immobilized with other carriers. The structure of the immobilized enzyme was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction. The D-tagatose synthesis reaction using the immobilized enzyme was optimized through both single-factor and statistical methods. The maximum yield of D-tagatose reached 30.12% from 1 M D-galactose at 68°C, with a productivity of 6.78 g/L/h. The immobilized enzyme was reusable for 20 cycles of reaction at 60°C, retaining over 55% of its initial activity and accumulating a high amount of 863.74 g/L D-tagatose. Furthermore, it efficiently converted guar gum hydrolysate into D-tagatose, retaining over 50% enzyme activity and yielding 114.32 g/L of D-tagatose after 10 cycles. The excellent enzyme reusability and the exploration of an economic substrate would significantly reduce production costs in large-scale D-tagatose synthesis in the future.
研究领域:
生物化学
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食品科学
✓ 已验证
产品关联
DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2024
·
IF 3.4
Dingyu Guo,Zhengchao Wang,Wanqing Wei,Wei Song,Jing Wu,Jian Wen,Guipeng Hu,Xiaomin Li,Cong Gao,Xiulai Chen,Liming Liu
D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency . Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds , and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; T m value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.
研究领域:
生物化学
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食品科学
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酶工程
✓ 已验证
DOI
PubMed
高影响因子
GREEN CHEMISTRY
·
2018
·Vol. 20
·期 10
·2382-2391
·
IF 8.586
Feng Sha,Yucong Zheng,Jiao Chen,Kequan Chen,Fei Cao,Ming Yan,Pingkai Ouyang
In this study, the bio-oxidation of galactitol to the valuable D-tagatose is presented. This proposed strategy could start with the refined by-product of waste xylose mother liquor formed by chemical hydrogenation. Through a computationally guided enzyme screening approach, a robust polyol dehydrogenase (PdPDH) was rapidly identified from a massive number of candidates. When coupled with a water-forming NADH oxidase (StNOX) for environmentally benign cofactor regeneration, subsequent reaction optimization facilitated the complete transformation of 100 g L−1 galactitol into the desired product within 15 h, with a yield of 91% in a 2 L scale preparative reaction. Compared to the current enzymatic isomerization system, our approach avoids low conversion, high operative temperatures and by-product formation, while enabling simplified product isolation.
研究领域:
生物技术
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酶学
·
可持续化学
✓ 已验证
产品关联
DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2020
·
IF 7.539
Guoyan Zhang,Hossain M. Zabed,Junhua Yun,Jiao Yuan,Yufei Zhang,Yang Wang,Xianghui Qi
In this study, a new strain of Lactobacillus plantarum (CY.6) was identified and its L-arabinose isomerase (L-AI) encoding gene ( ara A) was overexpressed in Escherichia coli BL21 for the biosynthesis of D-tagatose from milk whey powders (WP). Whole-cell biotransformation of lactose in WP into D-tagatose was done by three technological approaches, including 100%, 50% and 0% hydrolysis of lactose in WP before biotransformation, where simultaneous saccharification and biotransformation (SSB, 0% prior hydrolysis of lactose) produced maximum amounts of D-tagatose. Two-stage SSB provided 73.6% conversion efficiency (based on D-galactose) and 36.8% (in term of lactose), with 51.5 g/L of D-tagatose after 96 h, while concentration of D-tagatose produced after first stage was 34.4 g/L. Yield and volumetric productivity of D-tagatose after two-stage SSB were found to be 0.26 g/g of WP (0.37 g/g of lactose, 0.74 g/g of D-galactose produced from lactose) and 0.54 g/L/h, respectively.
研究领域:
生物技术
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微生物学
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食品工程
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酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2019
·
IF 6.669
Ganqiao Ran,Dan Tan,Jiping Zhao,Fan Fan,Qiang Zhang,Xingjuan Wu,Peiyao Fan,Xinlei Fang,Xiaoyun Lu
d -Allulose is a promising low-calorie sweetener especially for diabetes and obesity patients. The functionalized polyhydroxyalkanoate (PHA) nano-beads decorated with d -tagatose 3-epimerase (DTE) was produced in recombinant endotoxin-free ClearColi , whereby the expression, purification, and immobilization of the active DTE were efficiently combined into one step. The immobilized DTE exhibited remarkable enzyme activity of 649.3 U/g beads and extremely high stability at a harsh working condition (pH 7.0–8.0, 65 °C). When DTE-PHA beads were subjected to enzymatic synthesis of d -allulose, a maximum conversion rate of 33% can be achieved at pH 7.0 and 65 °C for 3 h, and DTE-PHA beads retained about 80% of its initial activity after 8 continuous cycles. Moreover, the d -allulose/ d -fructose binary mixture can be simply separated by a single cation exchange resin-equipped chromatography. Taken together, DTE-PHA beads are promising and robust nano-biocatalysts that will remarkably simplify the production procedures of d -allulose, contributing to its cost-effective production.
研究领域:
生物技术
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食品科学
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酶学
·
材料科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）