APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2022
·Vol. 106
·期 9
·3625-3637
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IF 5.56
Wang Juan,Qu Ge,Xie Leipeng,Gao Chao,Jiang Yingying,Zhang Yi-Heng P. Job,Sun Zhoutong,You Chun
Abstract Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. Key points • A screening method was established for the protein engineering of SsDHAD. • A R3 mutant of SsDHAD with 10-fold higher activity was obtained. • The R3 mutant exhibits higher productivity in the in vitro biosystems. Graphical abstract
研究领域:
酶学
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合成生物学
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生物催化
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ENZYME AND MICROBIAL TECHNOLOGY
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2025
·
IF 3.7
Haotian Cheng,Yuan Li,Yi Zhou,Shuke Wu,Yi-Heng P. Job Zhang
Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to glucose and α-D-glucose-1-phosphate. CBPs play an important role in the in vivo enzymatic utilization of cellulosic materials. Here we discovered a novel CBP from Thermoclostridium caenicola (TcCBP), displaying 50–75 % sequence homology with previously reported CBPs. Recombinant TcCBP was well expressed in E. coli BL21(DE3), with a 1.6-fold increase in soluble expression levels over the widely-used Clostridium thermocellum CBP (CtCBP). This enzyme exhibited broad pH adaptability, maintaining substantial activities across pH 4.0–7.5 in the synthetic direction and pH 5.0–7.5 in the phosphorolytic direction. Compared to CtCBP, TcCBP displayed the superior thermostability and a nearly 100-fold improvement in "Product-to-Enzyme Ratio" (PE value). The biosynthesis of myo -inositol from cellobiose was conducted by using this CBP along with other four thermophilic enzymes (i.e., phosphoglucomutase, inositol 1-phosphate synthase, inositol monophosphatase, and polyphosphate glucokinase) in one pot without step-by-step addition of enzymes. Approximately 96 mM of myo -inositol was produced from 50 mM of cellobiose. These results indicated that this enzyme could be a potential thermophilic enzyme used for the production of value-added biochemicals by in vitro BioTransformation.
研究领域:
酶工程
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分子生物学
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合成生物学
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代谢工程
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工业生物技术
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ENZYME AND MICROBIAL TECHNOLOGY
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2025
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IF 3.7
Xiaotong Kang,Hang Yuan,Weimeng Li,Yun Hu,Fubao Sun
Thermophilic endoglucanases are of big promise in enzymatic degradation of lignocellulosic substrate, yet their limited catalytic efficiency remains a major barrier to industrial application. This study focused on the improvement of activity and stability of thermostable endoglucanase Dt CelA from Dictyoglomus turgidum with a three-stage cascade engineering strategy that integrated model-guided site-directed mutagenesis, disulfide bond introduction, and carbohydrate-binding module (CBM) fusion. Among the variants, the triple mutant S25D/T265C/A305C fused with CBM2 (S25D/T265C/A305C-A-CBM2) exhibited a 90.56 % increase in specific activity toward CMC relative to the wild type, together with a 2.97-fold longer half-life at 75 °C and a 5.3-fold longer at 80 °C. This mutant also displayed 3.0- and 5.4-fold higher hydrolytic activity against Avicel and konjac glucomannan, respectively. On natural lignocellulosic substrates including filter paper, sugarcane bagasse, and corn stover, the mutant also showed potential application. The mutated endoglucanase Dt CelA was powerful with enhanced catalytic performance and thermostability after multicascade engineering, which can provide a robust framework for the rational design of cellulases that are optimized for high-temperature biorefinery process.
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FOOD CHEMISTRY
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2025
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IF 9.8
Jing Zhu,Yi Gao,Xingfei Li,Cheng Lu,Xing Zhou,Long Chen,Chao Qiu,Zhengyu Jin,Jie Long
Maltogenic α-amylase (MA, EC 3.2.1.133), which shows high thermal stability, can cleave long starch chains linked by α-1,4 glycosidic bonds. And glycogen branching enzyme (GBE, EC 2.4.1.18) can increase the branching degree of starch. At high temperatures, Elevated temperatures enhanced starch solubilization, improving the accessibility of both MA and GBE to glucan chains. Therefore, ungelatinized normal corn starch (NCS) emulsion (20 % w /w, dry basis) was first pretreated with α-amylase (MA) at 70 °C for 2 h, followed by enzymatic modification using the thermostable Pyrococcus horikoshii OT3 GBE mutants (H415W and T508K) for an additional 10 h modification. The physicochemical properties of the modified starch were determined by scanning electron microscopy and X-ray diffraction, etc. The results demonstrated that the dual enzyme treatment decreased rapid digestibility by 23.07 % and retrograde enthalpy by 5.51 J/g compared to NCS. This study provides guidance for the industrial enzymatic preparation of highly branched starch.
研究领域:
食品科学
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生物技术
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碳水化合物化学
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酶学
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淀粉加工
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高影响因子
Scientific Reports
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2017
·Vol. 7
·期 1
·1-10
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IF 4.259
Jia Xiaojing,Liu Ying,Han Yejun
Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65–70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn 2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h −1 , and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.
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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PubMed
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GREEN CHEMISTRY
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2024
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IF 9.3
Lin Fan,Shangshang Sun,Zhidan Zhang,Yanmei Qin,Peter Ruhdal Jensen,Chun You
In conjunction with the pressing global issue of climate change and the associated concern over global warming, increasing interest has emerged in the exploration of carbon dioxide (CO2) as a resource for the generation of a diverse array of products intended to serve societal needs. This study presents the development of an ATP-free and NAD-balanced in vitro synthetic enzymatic biosystem (ivSEB), which comprises only five cascade thermophilic enzymes, designed for the synthesis of L-malate through CO2 fixation powered by the utilization of D-glucose as a substrate. This designed ivSEB yields two moles of L-malate from one mole of D-glucose and two moles of CO2. Through meticulous refinement of reaction conditions and enzyme loading amounts, this ivSEB has demonstrated its capability to produce 6.85 mM of L-malate via CO2 fixation from an initial 5 mM of D-glucose with a molar product yield of 68.5%, and 2.45 mM of L-lactate as a byproduct. In the pursuit of assessing the industrial feasibility of this ivSEB, the study further subjected the system to the utilization of a high concentration (45.70 mM) of D-glucose. Although this endeavor necessitates additional optimization for enhanced efficiency, the present findings herald the emergence of an alternative avenue for the sustainable production of L-malate through CO2 fixation, thus bearing substantial promise for addressing ecological and industrial imperatives.
研究领域:
生物技术
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环境科学
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化学工程
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可持续化学
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酶学
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DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Yuvaraj Ravikumar,Abdullah Arsalan,Xinrui Tang,Guoyan Zhang,Xianghui Qi
l-Arabinose isomerase (l-AI) catalyzes d-galactose to produce the rare, industrially important sugar d-tagatose. Enzyme stability is vital for its application in industrial processes, and rational design-based protein engineering methods have been employed to improve its stability. This study employed molecular dynamics simulations (MDS)-guided rational redesign of flexible regions to improve the thermostability of mesophilic Bifidobacterium adolescentis l-AI (Ba-l-AI). Comparative MDS (200 ns, 340–360 K) of thermophilic l-AIs (Geobacillus kaustophilus Gk-l-AI and Thermotoga maritima Tm-l-AI) and mesophilic Ba-l-AI identified flexible regions in Ba-l-AI via RMSF and ionic interaction. Five stabilizing mutations (G78C, K46R, N187R, K112R, and N190R) were introduced to provide rigidness. MDS of the mutant revealed reduced RMSF/RMSD, improved compactness, and enhanced conformational stability across temperatures, supported by free-energy landscape analysis resembling thermophilic profiles. Experimentally, the mutant exhibited superior thermal stability (72 °C), broader pH/temperature tolerance, and efficient d-tagatose production. The redesigned Ba-l-AI’s rigid backbone and stability highlight its potential for industrial d-tagatose biosynthesis.
研究领域:
生物催化
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蛋白质工程
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结构生物学
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工业生物技术
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计算生物学
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酶学
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PubMed
高影响因子
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