Foods
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2022
·Vol. 11
·期 20
·3225
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IF 5.561
Xinrui Tang,Yingfeng An,Muhammad Waheed Iqbal,Hongri Cong,Guoyan Zhang,Yufei Zhang,Yuvaraj Ravikumar,Hossain M. Zabed,Mei Zhao,Haixing Zhou,Xianghui Qi
D-allulose is a natural rare sugar with important physiological properties that is used in food, health care items, and even the pharmaceutical industry. In the current study, a novel D-allulose 3-epimerase gene (Bp-DAE) from the probiotic strainBlautia producawas discovered for the production and characterization of an enzyme known as Bp-DAE that can epimerize D-fructose into D-allulose. Bp-DAE was strictly dependent on metals (Mn2+and Co2+), and the addition of 1 mM of Mn2+could enhance the half-life of Bp-DAE at 55 °C from 60 to 180 min. It exhibited optimal activity in a pH of 8 and 55 °C, and theKmvalues of Bp-DAE for the different substrates D-fructose and D-allulose were 235.7 and 150.7 mM, respectively. Bp-DAE was used for the transformation from 500 g/L D-fructose to 150 g/L D-allulose and exhibited a 30% of conversion yield during biotransformation. Furthermore, it was possible to employ the food-grade microbial speciesBacillus subtilisfor the production of D-allulose using a technique of whole-cell catalysis to circumvent the laborious process of enzyme purification and to obtain a more stable biocatalyst. This method also yields a 30% conversion yield.Keywords:D-allulose 3-epimerase;D-allulose;Blautia produca;probiotics;biochemical characterization;biotransformation
研究领域:
生物化学
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生物技术
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食品科学
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药学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Xinrui Tang,Abdullah Arsalan,Guoyan Zhang,Junhua Yun,Cunsheng Zhang,Xianghui Qi
D-Allose, a rare sugar, has gained significant attention not only as a low-calorie sweetener but also for its anticancer, antitumor, anti-inflammatory, antioxidant, and other pharmaceutical properties. Despite its potential, achieving high-level biosynthesis of D-allose remains challenging due to inefficient biocatalysts, low conversion rates, and the high cost of substrates. Here, we explored the food-grade coexpression of Blautia produca D-allulose 3-epimerase (Bp-DAE) and Bacillus subtilis L-rhamnose isomerase (BsL-RI) within a single cell using B. subtilis WB800N as the host. Using this system, D-allose was synthesized via a simple, cost-effective, one-pot enzymatic process, employing whole cells as catalysts and D-fructose as the substrate. The system exhibited optimal activity at 65 °C, pH 8.5, with 1 mM Mn2+ and 20 g/L of whole-cell dry weight. Initial production reached 12.5 g/L D-allose with a 12.5% yield from 100 g/L D-fructose. Optimization of dual promoter combinations enhanced production, achieving 15.0, 29.1, and 43.2 g/L D-allose from 100, 200, and 300 g/L D-fructose, with yields of 15.00, 14.55, and 14.40%, respectively. This D-allose production biocatalyst offers a scalable and economically viable platform for the industrial production of rare sugar.
研究领域:
生物化学
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食品科学
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工业微生物学
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产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2021
·
IF 3.493
Rong Wang,Xinqi Xu,Xuemei Yao,Hengtao Tang,Xin Ju,Liangzhi Li
Ribose-5-phosphate isomerase A (RpiA) is of great importance in biochemistry research, however its application in biotechnology has not been fully explored. In this study the activity of RpiA from Ochrobactrum sp. CSL1 (OsRpiA) towards D-allose was engineered based on sequential and structural analyses. Strategies of alanine scanning, rational design and saturated mutagenesis were employed to create three mutant libraries. A single mutant of K124A showed a 45 % activity improvement towards D-allose. The reaction properties of the mutant were analyzed, and a shift of optimal pH and higher thermal stability at low reaction temperatures were identified. The conversion of D-allose was also improved by 40 % using K124A, and higher activities on major substrates were found in the mutant’s substrate scope, implying its application potential in rare sugar preparation. Kinetics analysis revealed that K m of K124A mutant decreased by 12 % and the catalytic efficiency increased by 65 % towards D-allose. Moreover, molecular dynamics simulation illustrated the binding of substrate and K124A was more stable than that of the wild-type. The shorter distance and more relax bond angle between the catalytic residue of K124A and D-allose explained the activity improvement in detail. This study highlights the potential of OsRpiA as a biocatalyst for rare sugar preparation, and provides distinct evidences for its catalytic mechanism.
研究领域:
生物化学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2020
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IF 4.192
Xiaofeng Zhang,Xinqi Xu,Xuemei Yao,Rong Wang,Hengtao Tang,Xin Ju,Liangzhi Li
Ribose-5-phosphate isomerase B is of great importance for biocatalysis and biosynthesis, but the multifunctional residues in active sites hinder the research efforts. This study employed rational design strategies to locate the key residues of RpiB from Ochrobactrum sp. CSL1 (OsRpiB). A single-mutant S9T of a noncontact residue showed 80% activity improvement toward d-allose. A double-mutant S98H/S134H further increased the activity to 3.6-fold. The mutations were analyzed by kinetics and molecular dynamics analyses, indicating that S9T might enhance the substrate binding and catalysis by inducing a steric effect, and S98H/S134H could strengthen both ring opening and binding of d-allose. Though S98H/S134H showed low temperature stability, its potential was explored by isomerizing d-allose to d-psicose with higher conversion and in less reaction time. The findings of this study were beneficial for illustrating the complex functions of key residues in RpiBs and applying OsRpiB in preparing rare sugars.
研究领域:
生物化学
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分子生物学
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合成生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2024
·
IF 4.8
Han Zhang,Zhen Sun,Xinling Du,Qianqian Cheng,Fuchun Ji,Zishen Nie,Julei Zhan,Zhouping Wang,Aitao Li,Irina Delidovich,Yu Xia
d- Allulose is a rare sugar with multiple physiological functions whose application has been restricted by low conversion rate, weak thermostability of the used enzymes, and complex production. In this study, a simplified multienzyme cascade strategy was designed for biosynthesis of d- allulose from d- glucose, and a detailed investigation of the crucial enzyme allulose 6-phosphate phosphatase was conducted. Under optimized conditions, the yield of d- allulose reached 4.12 ± 0.14 and 80.20 ± 3.20 mmol/L using 10.0 and 200.0 mmol/L d- glucose as substrate, respectively. Furthermore, the influence of various factors on the cooling crystallization of d- allulose was investigated, and the crystallization process was optimized. Consequently, a crystallization recovery rate of 77.43% ± 2.50% and a purity of 98.70% ± 0.05% were achieved. Moreover, the obtained crystals of d -allulose were characterized by XRD, TGA-DSC, and optical microscopy.
研究领域:
生物化学
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食品科学
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产品关联
DOI
高影响因子
FOOD SCIENCE AND BIOTECHNOLOGY
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2023
·
IF 2.9
Zheng Qian,Long Si,Chen Zhi,Fu Jiaolong,Ju Xin,Li Liangzhi
Enzymatic preparation of rare sugars as an alternative to traditional sweeteners is an effective strategy to achieve a low-calorie healthy diet. Ribose-5-phosphate isomerase B (RpiB) is a key enzyme in the non-oxidative branch of the catalytic pentose phosphate pathway. Here, we investigated the potential of Curtobacterium flaccumfaciens ZXL1 ( C. flaccumfaciens ZXL1) derived RpiB (CfRpiB) in D-allose preparation. The optimal reaction conditions for recombinant CfRpiB were found experimentally to be pH 7.0, 55 °C, and no metal ions. The kinetic parameters K m , k cat , and catalytic efficiency k ca t / K m were 320 mM, 4769 s −1 , and 14.9 mM −1 s −1 respectively. The conversion of D-allulose by purified enzyme (1 g L −1 ) to D-allose was 13% within 1 h. In addition, homology modeling and molecular docking were used to predict the active site residues: Asp13, Asp14, Cys72, Gly73, Thr74, Gly77, Asn106, and Lys144.
研究领域:
生物化学
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生物技术
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食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2021
·
IF 7.514
Dan Bu,Zong-cai Tu,Hui Wang,Yue-ming Hu,Qing Sun,Guang-xian Liu
The mechanism of d -allose in reducing the allergenicity and digestibility of ultrasound-pretreated α-lactalbumin (α-LA) was studied. The intensity reduction and peak red shift occurred in fluorescence spectra of glycated samples. Enzyme-linked immunosorbent assay and basophil degranulation analysis showed that d -allose significantly reduced the allergenicity of α-LA, and ultrasound-pretreated α-LA showed the lowest allergenicity after glycation. Compared with α-LA, the degree of hydrolysis decreased in glycated samples. Size-exclusion high-performance liquid chromatography showed that the glycated α-LA was resistant to digestive enzymes. The glycated sites and average degree of substitution per peptide molecule were determined using LC Orbitrap MS/MS. These results suggested that the masking of linear allergenic epitopes by glycation could reduce the allergenicity. Therefore, the combination of ultrasound pretreatment and glycation is a potential method to reduce protein allergenicity in food processing and provides a useful approach for the application of rare sugars in food processing.
研究领域:
食品科学
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过敏学
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质谱学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2024
·
IF 4.8
Beini Chen,Xinrui Tang,Yufei Zhang,Hossain M. Zabed,Yuvaraj Ravikumar,Muhammad Waheed Iqbal,Jiayin Wang,Mei Zhao,Xianghui Qi
d -Allulose, is an improtant rare sugar, having health-promoting properties and applications in the food, pharmaceutical and nutrition sectors. White the aldol reaction-based method shows promise as an alternative to the “Izumori” strategy for ᴅ-allulose bioproduction, its industrial exploitability is hindered by the low product yield in reaction equilibrium and the high costs associated with the use of purified enzymes . To address these limitations, this study explored cell-based biosynthesis of ᴅ-allulose from low-cost glycerol by overexpression the enzymes involved in the ᴅ-allulose aldol condensation (AC) biosynthesis pathway, namely fructose-1-phosphatase (YqaB), alditol oxidase (AldO), and l -rhamnulose-1-phosphate aldolase (RhaD), in Escherichia coli . Results indicated that RhaD is a rate-limiting step of the ᴅ-allulose biosynthetic pathway. Then, biosensor-based high throughput screening was employed for the directed evolution of RhaD. In this way, two RhaD mutants, namely RhaD N156D and RhaD P184S , were successfully screened from 40,129 individual colonies, which exhibited respectively achieved a ᴅ-allulose titer of 3.1 g/L and 2.9 g/L, respectively in shake flasks, representing a more than 20% improvement compared to the wildtype strain. Overall, the ᴅ-allulose biosensor-assisted key enzyme evolution strategy and the high throughput screening method presented in this work provide a paradigm for improving rare sugars’ high-valued productions from cheap substrates.
研究领域:
生物技术
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代谢工程
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酶工程
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合成生物学
✓ 已验证
产品关联
DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2023
·
IF 3.4
Shuangshuang Duan,Yonghua Chen,Guodong Wang,Zebin Li,Shitong Dong,Yingshuai Wu,Yuanwei Wang,Chunling Ma,Ruiming Wang
D -Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L -Rhamnose isomerase ( L -Rhi) is the earliest enzyme found to catalyze the production of D -allose from D -psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D -allose. In this study, L -Rhi derived from Bacillus subtilis was employed as the research subject, and D -psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure , tertiary structure , and interactions with ligands of the enzyme. The yield of D -allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D -allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively. According to modeling analysis, manganese (Mn 2+ ) had no significant effect on the production of D -psicose from D -psicose by L -Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D -psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D -psicose and facilitating its conversion to D -allose, providing the basis for the production of D -allose.
研究领域:
生物化学
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酶学
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生物技术
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结构生物学
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产品关联
DOI
PubMed
高影响因子
ACS Food Science & Technology
·
2021
·
IF 0
Xiaoyan Chen,Fan Wang,Shijie Liu,Qiang Yu,Wen Wang,Yu Zhang,Zhongming Wang,Zhenhong Yuan
The synthesis efficiency of d-allulose, an ideal sugar substitute and dietary supplement, from d-glucose with whole-cell catalysts was improved by three out of the four tested deep eutectic solvents (DESs) based on choline chloride (ChCl). The promotion effects on the product yields with increments of up to 2.6-fold were observed in reactions with the presence of DESs in the order ChCl/glycerol < ChCl/urea < ChCl/ethylene glycol. DES compositions displayed distinct influences, but all positive effects appeared in the reactions with low DES contents (1–10%). Higher DES contents produced negative effects, and 40% even totally suppressed the reaction. The enhancement of efflux of intracellular molecules was proved by an analysis of the nucleic acid and protein contents in the medium. DES assisted in the increase in cell membrane permeability rather than in the damage of cell wall integrity, accelerating quick contacts between the enzyme and substrate and improving the conversion rate.
研究领域:
化学工程
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生物化学
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食品科学
✓ 已验证
DOI
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
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材料科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Chemistry
·
2020
·
IF 3.693
Wang Yang,Ravikumar Yuvaraj,Zhang Guoyan,Yun Junhua,Zhang Yufei,Parvez Amreesh,Qi Xianghui,Sun Wenjing
D-allulose, which is one of the important rare sugars, has gained significant attention in the food and pharmaceutical industries as a potential alternative to sucrose and fructose. Enzymes belonging to the D-tagatose 3-epimerase (DTEase) family can reversibly catalyze the epimerization of D-fructose at the C3 position and convert it into D-allulose by a good number of naturally occurring microorganisms. However, microbial synthesis of D-allulose is still at its immature stage in the industrial arena, mostly due to the preference of slightly acidic conditions for Izumoring reactions. Discovery of novel DTEase that works at acidic conditions is highly preferred for industrial applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on D-fructose at pH 6.0 and 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on -fructose at pH 6.0 and 50°C with a Kcat/Km value of 45 mM−1min−1. The 500 g/L D-fructose, which corresponded to 30% conversion rate. With these interesting catalytic properties, this enzyme could be a promising candidate for industrial biocatalytic applications.
研究领域:
食品科学
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生物化学
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酶学
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微生物学
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）