BIORESOURCE TECHNOLOGY
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2025
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IF 9
Jingwen Zhang,Binge Zhang,Feiyu Duan,Zehui Xuan,Tong Sun,Lili Lu
β-Galactosidases are important enzymatic tools for glycosylation, but their properties vary greatly with the source. Here, ten putative β-galactosidase genes, designated as bga 1 to bga 10, encoding proteins Bga1 to Bga10, were mined from an environmental metagenomic dataset comprising 119,152 sequences. Five of the encoded enzyme proteins exhibited less than 80% sequence similarity to known enzymes, but displayed conserved catalytic sites in their predicted three-dimensional models. After heterologous expression and characterization, two recombinant enzymes showed specific hydrolysis activity toward o -nitrophenyl-β- d -galactopyranoside. One of them, Bga4R, exhibited remarkable activity at pH 7.4 and 50℃, with excellent alkaline stability. Notably, Bga4R tolerated a wide range of acceptors for transglycosylation. It catalyzed galactosyl transfer to various monosaccharides and sugar alcohols, and enabling the synthesis of diverse glycosylated derivatives. This study identifies a novel GH 1 β-galactosidase as a powerful tool for glycosylation engineering, with promising potential for synthesizing galactosides valuable to food and pharmaceutical industries.
研究领域:
生物化学
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微生物学
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合成生物学
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PubMed
高影响因子
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
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2021
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IF 3.346
Xue Yemin,Xue Mengke,Xie Fang,Zhang Mengchen,Zhao Hongyang,Zhou Tao
Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a β-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-β-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-β-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-β-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.
研究领域:
生物化学
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酶工程
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INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY
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2017
·Vol. 52
·期 12
·2596-2603
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IF 1.64
Lin Ge,Jingcong Xie,Tao Wu,Shanshan Zhang,Linguo Zhao,Gang Ding,Zhenzhong Wang,Wei Xiao
Summary A novel α-L-rhamnosidase was isolated and purified from Aspergillus oryzae NL-1. The enzyme was purified 13.2-fold by ultrafiltration, ion exchange and gel filtration chromatography with an overall recovery of 6.4% and specific activity of 224.4 U/mg, and the molecular mass of its subunit was approximately 75 kDa. Its optimal temperature and pH were 65 °C and 4.5, respectively. The enzyme was stable in the pH range 3.5–7.0, and it showed good thermostability at higher temperatures. The K M , kcat and kcat/K M values were 5.2 m m , 1624 s −1 and 312 s −1 m m −1 using p NPR as substrates, respectively. Moreover, the enzyme exhibited transglycosylating activity, which could synthesise rhamnosyl mannitol through the reactions of transglycosylation with inexpensive rhamnose as the glycosyl donor. Our findings indicate that the enzyme has potential value for glycoside synthesis in the food industry.
研究领域:
生物化学
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食品科学
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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2020
·Vol. 104
·期 11
·4927-4943
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IF 3.53
Deng Pengjun,Meng Chunyu,Wu Yan,Xu Jie,Tang Xiaoqing,Zhang Xuecheng,Xiao Yazhong,Wang Xiaotang,Fang Zemin,Fang Wei
A novel β -glucosidase, BglD1 with high β -galactosidase and transglycosidation activities, was screened and cloned from the deep-sea bacterium Bacillus sp. D1. BglD1 exhibited the maximal β -glucosidase and β -galactosidase activities at 55–60 °C and pH 5.5–6.0. The enzyme maintained approximately 50% of its original activity at 35 °C and pH 6.0 after 120-h incubation. When applied to synthesize galacto-oligosaccharides (GOS), BglD1 generated 118.3 g/L GOS (33.8% (w/w)) from 350 g/L lactose, with trisaccharide Gal- β (1 → 3)-Lac and disaccharide Gal- β (1 → 4)-Gal as the main components. Furthermore, BglD1 could hydrolyze lactose in milk and produce GOS simultaneously. Using milk as the substrate, BglD1 hydrolyzed 88.5% lactose and produced 3.3 g/L GOS after incubation at 30 °C for 1 h. To improve the transglycosidation activity, a mutant BglD1:E224T was generated based on the semi-rational design. The GOS yield of BglD1:E224T was 11.5% higher than that of BglD1 when using lactose solution as the substrate. Thus, BglD1 and the mutant could be used as beneficial alternatives of the existing β -galactosidases for the production of GOS.
研究领域:
微生物学
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酶工程
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食品科学
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Molecular Catalysis
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2024
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IF 3.9
Qiqi Xing,Xiaoyu Wang,Qi Li,Changning Lu,Yi Wu,Jianjun Pei,Linguo Zhao
β-Galactosidase is a crucial class of biocatalysts, renowned for its ability to catalyze the hydrolysis of non-reducing terminal β-d-galactoside bonds, while also demonstrating significant potential in galactosylation synthesis reactions. In this study, a novel GH1 β-galactosidase gene VthBga was screened and cloned from Vulcanisaeta thermophila . Soluble expression of the VthBga was induced at 47 °C in Escherichia coli BL21. VthBga exhibited optimal activity at 95 °C and pH 4.5 with p -nitrophenyl-β-d-galactoside ( p NPGal) as a substrate and displayed excellent thermotolerance below 90 °C. In addition, VthBga possessed not only a diverse array of substrate hydrolytic activities but also inherent galactosylation activity, rendering it a promising candidate for the galactosylation of the natural compound. To enhance its transglycosylation activity, a superior mutant Vth-A266G/F441T was obtained by adjusting the transglycosylation/hydrolysis ratio through a semi-rational design. Under optimal conditions, this mutant produced 44.5 % tyrosol galactoside, representing a significant 2.3-fold increase compared to the wild-type strain, highlighting its practical value in related biosynthesis applications.
研究领域:
生物化学
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分子生物学
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酶工程
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FOOD CHEMISTRY
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2025
·
IF 9.8
Zhuoning Cao,Chengqiang Li,Hong Jiang,Francesco Secundo,Xiangzhao Mao
β- N -acetylhexosaminidases garnered attention in the enzymatic synthesis of lacto- N -triose II (LNT2) as the backbone precursor of human milk oligosaccharides (HMOs). In this study, β- N -acetylglucosaminidases Hex(Sv)-2557 from Streptomyces violascens ATCC 27968 was engineered based on a stabilizing intermediate strategy to improve its transglycosylation activity for LNT2 synthesis. A mutant Hex(Sv)-2557(D297K) with a transglycosylation activity of 38.4 U/mg with p NP-GlcNAc −1.9-fold higher than that of Hex(Sv)-2557– was obtained and characterized. Instead, the hydrolase activity of the mutant was 73 % lower compared to the wild-type enzyme. Importantly, the mutant can use N -acetyl chitobiose (GlcNAc 2 ) as the donor for LNT2 synthesis. The LNT2 yield of 14.85 % was obtained when the synthetic reaction, catalyzed by the mutant Hex(Sv)-2557(D297K), started from whey powder and GlcNAc 2 -prepared from chitin by chitinase ChiA and ChiB. This study has altered the donor for the action by directed modification and promoting the high-value utilization of whey powder and chitin.
研究领域:
生物化学
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酶工程
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食品科学
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Chengqiang Li,Zhuoning Cao,Hong Jiang,Francesco Secundo,Xiangzhao Mao
β-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family β-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-β-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 β-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.
研究领域:
生物化学
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酶学
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食品科学
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生物技术
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高影响因子
Biotechnology for Biofuels
·
2020
·Vol. 13
·期 1
·1-14
·
IF 4.815
Zhang Alei,Mo Xiaofang,Zhou Ning,Wang Yingying,Wei Guoguang,Chen Jie,Chen Kequan,Ouyang Pingkai
Background N -Acetyl glucosamine (GlcNAc) and N -Acetyl chitooligosaccharides ( N -Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N -Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc) 4 –(GlcNAc) 7 ) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β- N -acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N -Acetyl COSs. Results The gene encoding the novel β- N -acetyl glucosaminidase, designated Cm NAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of Cm NAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The Cm NAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified Cm NAGase toward p -nitrophenyl- N -acetyl glucosaminide ( p NP-GlcNAc) was 4878.6 U/mg of protein. Cm NAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max , K m , K cat , and K cat / K m of Cm NAGase for p NP-GlcNAc were 16,666.67 μmol min −1 mg −1 , 0.50 μmol mL −1 , 25,555.56 s −1 , and 51,111.12 mL μmol −1 s −1 , respectively. Analysis of the hydrolysis products of N -Acetyl COSs and colloidal chitin revealed that Cm NAGase is a typical exo-acting NAGase. Particularly, Cm NAGase can synthesize higher N -Acetyl COSs ((GlcNAc) 3 –(GlcNAc) 7 ) from (GlcNAc) 2 –(GlcNAc) 6 , respectively, showed that it possesses transglycosylation activity. In addition, Cm NAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions The observations recorded in this study that Cm NAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N -Acetyl COSs.
研究领域:
微生物学
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生物化学
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酶工程
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Synthetic and Systems Biotechnology
·
2025
·
IF 4.4
Dandan Feng,Tianshu Zhang,Guan Zhou,Yanli Cao,Quan Luo,Huifang Xu,Xuefeng Lu
2′-Deoxy-2′-fluoroadenosine (2′-F-dA) is a nucleoside analogue used as a key building block for oligonucleotide drugs. It can be biosynthesized from a low-cost 2′-deoxy-2′-fluorouridine via one-pot transglycosylation catalyzed by a thymidine phosphorylase (TP) and a purine nucleoside phosphorylase (PNP). However, reliance on purified enzymes and low space-time yields present challenges for industrial application of the process. Here, we develop a whole-cell-based biocatalytic system employing TP and PNP from Escherichia coli , which demonstrates high catalytic efficiency and operational simplicity in scaled-up reaction. In particular, a thermal pretreatment of TP- and PNP-expressing whole cells, determined as 50 °C for 3 h, effectively suppressed endogenous deamination side reaction while enhancing 2′-F-dA yield. Subsequent optimization of enzyme and substrate loadings and their relative ratios achieved an unprecedented space-time yield of 1.22 g/L/h with 88.1 g/L product titer in a 500 mL scaled-up reaction, manifesting a highest total conversion of 68.2 %. An integrated purification process yielded gram-scale solid powder of 2′-F-dA with 98.0 % chemical purity and 85.0 % recovery. This novel whole-cell biocatalytic process demonstrates significant industrial potential for the production of 2′-F-dA.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2019
·
IF 3.571
Yao Li,Lan-hua Liu,Xiao-qin Yu,Yu-xin Zhang,Jing-wen Yang,Xue-qin Hu,Hong-bin Zhang
Bioglycosylation is an efficient strategy to improve the biological activity and physicochemical properties of natural compounds for therapeutic drug development. In this study, two caffeic acid phenethyl ester (CAPE) glucosides (G-CAPE and 2G-CAPE) were synthesized by transglycosylation with dextransucrase from Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor. The products were purified and the structures were characterized. The physicochemical properties, anti-inflammatory activity, and cytotoxicity of the two CAPE glucosides were measured. The water solubility of G-CAPE and 2G-CAPE is 35 and 90 times higher, respectively, than that of CAPE. Compared to CAPE, the monoglycoside product showed superior anti-inflammatory effects, and its inhibition rate of NO, IF-6, and TNF-α is 93.4%, 76.81%, and 56.58% in RAW 264.7 macrophages, respectively, at 20 μM. Also, the cytotoxicity of both products was significantly improved. These glycosylation-modified CAPEs circumvent some of the flaws in CAPE application in anti-inflammatory drugs.
研究领域:
药学化学
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生物化学
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药理学
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高影响因子
危险品化学品经营许可证（带存储）