Foods
·
2024
·Vol. 13
·期 11
·1777
·
IF 4.7
Xianwen Ma,Dian Zou,Anying Ji,Cong Jiang,Ziyue Zhao,Xiaoqi Ding,Zongchen Han,Pengfei Bao,Kang Chen,Aimin Ma,Xuetuan Wei
In this study, a novel strain for degrading chitin was identified asBacillus paralicheniformisHL37, and the key chitinase CH1 was firstly mined through recombinant expression inBacillus amyloliquefaciensHZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes inB. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1.
研究领域:
微生物学
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生物化学
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分子生物学
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PubMed
高影响因子
Biocatalysis and Agricultural Biotechnology
·
2019
·
IF 0
Feng-Qin Gong,Qi-Shun Liu,Hai-Dong Tan,Tang Li,Cheng-Yu Tan,Heng Yin
Acetoin (3-hydroxy-2-butanone) is an important four-carbon compound widely used in the food industry and other industrial applications. This study aimed to identify a novel butanediol dehydrogenase that can efficiently catalyze the formation of acetoin. A novel butanediol dehydrogenase, Bt BDH, was identified from Bacillus thuringiensis subsp. Kurstaki ACCC 10066, and its enzymatic properties were characterized. The optimum pH and temperature for the oxidation activity of Bt BDH were 10.0 and 50 °C, respectively, and those for the reduction activities were 7.5 and 35 °C, respectively. In addition, it exhibits stability over a wide pH range (6–10) and temperatures up to 70 °C. Bt BDH showed good stability after storage for 3 months at 4 °C. Moreover, ethylenediaminetetracetic acid (EDTA) inhibits the enzymatic activity of Bt BDH, indicating that the enzyme is metal-dependent. This study characterized a novel (2R,3R) −2,3-butanediol dehydrogenase. Its excellent oxidation activity and stability ensure its great industrial application potential in the production of acetoin.
研究领域:
生物化学
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微生物学
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酶学
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DOI
BIOCHEMICAL JOURNAL
·
2023
·Vol. 480
·期 21
·1753-1766
·
IF 4.1
Cui Mengyu,Wei Yifeng,Tan Jason,Li Tong,Jiao Xinan,Zhou Yan
Phloroglucinol (1,3,5-trihydroxybenzene) is an important intermediate in the degradation of flavonoids and tannins by anaerobic bacteria. Recent studies have shed light on the enzymatic mechanism of phloroglucinol degradation in butyrate-forming anaerobic bacteria, including environmental and intestinal bacteria such as Clostridium and Flavonifractor sp. Phloroglucinol degradation gene clusters have also been identified in other metabolically diverse bacteria, although the polyphenol metabolism of these microorganisms remain largely unexplored. Here, we describe biochemical studies of polyphenol degradation enzymes found in the purple non-sulfur bacterium Rubrivivax gelatinosus IL144, an anaerobic photoheterotroph reported to utilize diverse organic compounds as carbon sources for growth. In addition to the phloroglucinol reductase and dihydrophloroglucinol cyclohydrolase that catalyze phloroglucinol degradation, we characterize a Mn2+-dependent phloretin hydrolase that catalyzes the cleavage of phloretin into phloroglucinol and phloretic acid. We also report a Mn2+-dependent decarboxylase (DeC) that catalyzes the reversible decarboxylation of 2,4,6-trihydroxybenzoate to form phloroglucinol. A bioinformatics search led to the identification of DeC homologs in diverse soil and gut bacteria, and biochemical studies of a DeC homolog from the human gut bacterium Flavonifractor plautii demonstrated that it is also a 2,4,6-trihydroxybenzoate decarboxylase. Our study expands the range of enzymatic mechanisms for phloroglucinol formation, and provides further biochemical insight into polyphenol metabolism in the anaerobic biosphere.
研究领域:
微生物学
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生物化学
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环境科学
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DOI
PubMed
高影响因子
Foods
·
2022
·Vol. 11
·期 22
·3722
·
IF 5.561
Hongbin Wang,Yuanfu Zhang,Zhaoting Yuan,Xiaotong Zou,Yuan Ji,Jiayi Hou,Jinfang Zhang,Fuping Lu,Yihan Liu
Until now,Streptoverticillium mobaraensetransglutaminase (TG) is the only commercialized TG, but limited information is known about its selection tendency on crosslinking sites at the protein level, restricting its application in the food industry. Here, four recombinantBacillusTGs were stable in a broad range of pH (5.0–9.0) and temperatures (<50 °C), exhibiting their maximum activity at 50–60 °C and pH 6.0–7.0. Among them, TG ofB. cereus(BCETG) demonstrated the maximal specific activity of 177 U/mg. A structural analysis indicated that the Ala147-Ala156 region in the substrate tunnel of BCETG played a vital role in catalytic activity. Furthermore, bovine serum albumin, as well as nearly all protein ingredients in soy protein isolate and whey protein, could be cross-linked by BCETG, and the internal crosslinking paths of three protein substrates were elucidated. This study demonstratedBacillusTGs are a candidate for protein crosslinking and provided their crosslinking mechanism at the protein level for applications in food processing.Keywords:transglutaminase;enzymatic characterization;structural analysis;crosslinking properties;protein substrate crosslinking mechanism
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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PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
·
2022
·
IF 3.141
YuLiang Jiao,YuYing Zhu,ShuMin Zeng,ShuFang Wang,Jing Chen,XiangHong Zhou,GuiZhen Ma
A novel uricase producing marine bacterium Priestia flexa alkaAU was isolated and identified. The 16S rDNA and the uricase coding gene were sequenced, analyzed and submitted to GenBank. The uricase from Priestia flexa alkaAU (PFU) was purified, determined to be 58.87 kDa, and conjugated with carboxymethyl chitosan (CMCS) by ionic gelation. CMCS conjugation had no effect on the optimum pH of PFU but decreased the optimum temperature by 10 °C. CMCS conjugation increased the specific activity of PFU by 53% at the human body temperature (37 °C) and small intestine’s pH (pH 6.8). Uricase thermostabilizing ability of CMCS was significant in the range of 37–80 °C but not at lower temperatures. For improvement of the pH stability of PFU, CMCS was more effective at pHs 3–5 than pHs 6–11. CMCS increased the half-life of PFU against artificial intestinal fluid by 1.5 folds, which demonstrated the potential capability of CMCS–PFU for oral administration.
研究领域:
海洋微生物学
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酶学
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生物技术
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药学
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DOI
PubMed
高影响因子
Journal of Integrative Agriculture
·
2018
·
IF 1.042
Zhen-gang LIU,Huan-huan WANG,Chao-bin XUE
Laccase (EC 1.10.3.2) is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction, which generally includes two types of laccase, Lac1 and Lac2. Lac1 oxidizes toxic compounds in the diet, and Lac2 is known to play an important role in melanizing the insect exoskeleton. In this study, we cloned and sequenced the cDNA of the diamondback moth, Plutella xylostella Lac2 ( PxLac2 ), from the third instar larvae using polymerase chain reaction (PCR) and rapid amplification of cDNA ends techniques. The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp. PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa. Moreover, we determined the expression levels of PxLac2 in different stages by quantitative PCR (qPCR). The results indicated that PxLac2 was expressed differently in different stages. We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae. We also investigated the enzymatic properties of laccase, which had optimal activity at pH 3.0 and at 35°C. Under these optimal conditions, laccase had a Michaelis constant ( K m ) of 0.97 mmol L −1 , maximal reaction speed ( V m ) of 56.82 U mL −1 , and activation energy ( E a ) of 17.36 kJ mol −1 to oxidize 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt). Type II copper enhanced laccase activity below 0.8 mmol L −1 and reduced enzyme activity above 0.8 mmol L −1 with an IC 50 concentration of 1.26 mmol L −1 . This study provides insights into the biological function of laccase.
研究领域:
昆虫学
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分子生物学
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DOI
高影响因子
STARCH-STARKE
·
2025
·
IF 2.7
Zixuan Lin,Tingting Hu,Lin Lin,Tao Li,Yan Yan,Wei Wei,Dongzhi Wei
Although α-amylases are critical biocatalysts in starch processing, fungal α-amylase diversity remains largely untapped. This study presents the first systematic characterization of an α-amylase from Penicillium brevicompactum NRRL864. Maximum α-amylase activity of 195 U/mL was obtained at pH 6.0 and 55°C under optimized fermentation conditions. These conditions consisted of 0.75% starch, 3% peptone, 0.5% NaCl, incubation at 20°C for 7 days. A novel α-amylase gene (DF) was identified via phylogenetic analysis and heterologously expressed in Escherichia coli . The gene encodes a 551-amino-acid α-amylase with an open reading frame of 1656 bp, showing 68.36% sequence identity to the α-amylase from Penicillium expansum . The DF enzyme displayed a specific activity of 489.4 U/mg under optimal conditions of 50°C and pH 6.0, and a 124.2% activity enhancement with 1 mM Ca 2 ⁺, indicating Ca 2 ⁺-dependence. The activity of DF was inhibited by a range of chemical reagents, including EDTA and urea. Substrate specificity assays confirmed that the DF enzyme exhibited optimal activity in hydrolyzing soluble starch. DF-mediated saccharification of corn starch yielded a reducing sugar concentration of 317.9 µg/mL within 70 min, confirmed by thin-layer chromatography. Beyond identifying P. brevicompactum as a novel α-amylase source, this work demonstrates DF as a Ca 2 ⁺-responsive GH13 enzyme with significant potential for industrial applications in biofuel and sweetener production.
研究领域:
生物技术
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酶学
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微生物学
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工业生物催化
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分子生物学
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高影响因子
GLYCOCONJUGATE JOURNAL
·
2018
·Vol. 35
·期 3
·255-263
·
IF 2.548
Guo Bi-Shan,Zheng Feng,Crouch Lucy,Cai Zhi-Peng,Wang Meng,Bolam David N.,Liu Li,Voglmeir Josef
A putative GH35 β-galactosidase gene from the mucin-degrading bacterium Akkermansia muciniphila was successfully cloned and further investigated. The recombinant enzyme with the molecular mass of 74 kDa was purified to homogeneity and biochemically characterised. The optimum temperature of the enzyme was 42 °C, and the optimum pH was determined to be pH 3.5. The addition of sodium dodecyl sulphate (SDS) reduced the enzyme’s activity significantly. The addition of Mg 2+ -ions decreased the activity of the β-galactosidase, whereas other metal ions or EDTA showed no inhibitory effect. The enzyme catalysed the hydrolysis of β1,3- and β1,6- linked galactose residues from various substrates, whereas only negligible amounts of β1,4-galactose were hydrolysed. The present study describes the first functional characterisation of a β-galactosidase from this human gut symbiont.
研究领域:
微生物学
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生物化学
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DOI
PubMed
高影响因子
Processes
·
2023
·Vol. 11
·期 1
·255
·
IF 3.5
Ke Chen,Jingyun Zhang,Xing Liu,Peiyuan Zhang,Wenlong Yue,Zhiqiang Cai
Aspergillussp. D-23 was obtained by ultraviolet-diethyl sulfate (UV-DES) compound mutagenesis fromAspergillussp. C18 that the α-galactosidase was purified from. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the purified enzyme demonstrated apparent homogeneity. The monomeric α-galactosidase’s native molecular weight was 125 kDa. The optimal temperature of α-galactosidase was 65 °C, and 75% of the initial enzyme activity could be maintained between 45 and 55 °C. Its optimal pH was 5.0 with good pH stability. After incubating for 2 h at pH 3.0–8.0, it could retain more than 80% of its original activity. Different concentrations of metal ions had different effects on the α-galactosidase activity. High concentrations of Cu2+could strongly inhibit enzyme activity and low concentrations of Fe2+could promote enzyme activity. Additionally, as shown by thin layer chromatography and high-performance liquid chromatography, the enzyme also had good hydrolysis ability, which could efficiently hydrolyze melibiose and raffinose by more than 95%. Therefore, these excellent characteristics could make α-galactosidase a good candidate for the food and feed industries.Keywords:α-galactosidase;enzymatic characterization;Aspergillussp.;enzymatic hydrolysis;higher molecular weight
研究领域:
生物化学
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食品科学
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微生物学
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高影响因子
MOLECULES
·
2022
·Vol. 27
·期 15
·4720
·
IF 4.927
Xinjun Yu,Zengchao Jiang,Xiaodan Xu,Changyi Huang,Zheyi Yao,Xiao Yang,Yinjun Zhang,Dongsheng Wang,Chun Wei,Xuwei Zhuang
Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase fromOstrinia furnacalis(OfHex1), endo-chitinase fromTrichoderma viride(TvChi1), and multifunctional chitinase fromChitinolyticbacter meiyuanensis(CmChi1), were expressed in thePichia pastorissystem, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.Keywords:chitinase;shrimp and crab shell;N-acetylglucosamine;mechanoenzymology;heterologous expression
研究领域:
生物化学
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生物技术
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环境科学
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PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2012
·Vol. 97
·期 3
·1113-1120
·
IF 3.425
Jia Dongxu,Li Jianghua,Liu Long,Zhang Dongxu,Yang Yu,Du Guocheng,Chen Jian
A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris . The fragment was then amplified by polymerase chain reaction and inserted into the site between Eco RI and Not I sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae . The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using Sal I and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca 2+ and Mg 2+ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant ( K
m ) of 1.89 mg/mL and a maximum reaction rate ( V
max ) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris , and the achieved PVADH yield is the highest ever reported.
研究领域:
生物化学
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分子生物学
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微生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Zhongyu Shi,Xiasen Wei,Yunfan Wei,Zheyi Zhang,Sibao Wan,Haiyan Gao,Zhen Qin
A novel glycoside hydrolase (GH) family 16 multi-domain β-1,3-1,4-glucanase (FsGlc16A) from Fibrobacter sp. UWP2 was identified, heterogeneously expressed, and its enzymatic properties, protein structure and application potential were characterized. Enzymological characterization showed that FsGlc16A performed the optimal catalytic activity at pH 4.5 and 50 °C with a specific activity of 3263 U/mg. FsGlc16A exhibited the substrate specificity towards oat β-glucan, barley β-glucan and lichenan, and in addition, it hydrolyzed oat β-glucan and lichenan into different β-glucooligosaccharides with polymerization degrees of 3–4, which further illustrated that it belonged to the endo-type β-1,3-1,4-glucanase. FsGlc16A was classified in subfamily25 of GH16. A ‘PXSSSS’ repeats domain was identified at the C-terminus of FsGlc16A, which was distinct from the typical GH family 16 β-1,3-1,4-glucanases. Removing the ‘PXSSSS’ repeats domain affected the binding of the substrate to FsGlc16A and reduced the enzyme activity. FsGlc16A displayed good potential for the applications, which hydrolyzed oat bran into β-glucooligosaccharides, and reduced filtration time (18.89 %) and viscosity (3.64 %) in the saccharification process. This study investigated the enzymatic properties and domain function of FsGlc16A, providing new ideas and insights into the study of β-1,3-1,4-glucanase.
研究领域:
生物化学
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酶学
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微生物学
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食品科学
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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）