JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
IF 6.2
Pengfei Li,Lin Kong,Shihan Yang,Haonan Chen,Xiumei Wang,Lixia Pan,Mingbo Qu
Chitinases that play pivotal roles in chitin biodegradation for high-value bioconversion often adopt multimodular architectures with catalytic and binding domains connected by linkers. The linkers have been proven to possess functional roles, yet their mechanistic contributions remain poorly defined. In this study, a systematic analysis of 1242 bacterial glycoside hydrolase family 18 chitinases revealed a preferred linker length of 30–45 amino acids and an enrichment in threonine–proline (TP) motifs. Engineered Serratia marcescensSmChiC variants with controlled lengths (15–60 residues) demonstrated that a 45-residue linker (GS9) optimally enhances hydrolytic efficiency (22.4–39.8% activity increase) across α-/β-/colloidal chitins, with QCM-D confirming enhanced substrate binding/hydrolysis dynamics. Strikingly, engineered SmChiC variants with different rigidities demonstrated that the TP-linker variant outperformed the wild-type, flexible (GS9), and rigid (EK) variants, exhibiting rapid substrate association as revealed by HS-AFM. These findings highlight the critical role of linker design in optimizing chitinase function and provide a foundation for engineering enhanced glycoside hydrolases.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2026
·
IF 6.7
Xingyue Wang,Jianrong Chen,Liyan Yang,Zhong Cheng,Dengfeng Yang,Huazhou Chen,Lixia Pan
Engineering chitinases with simultaneous improvements in catalytic efficiency and thermostability remains challenging. Here, we present a computational strategy targeting the insertion domain (CID), a noncatalytic auxiliary region. Using CaChi18A_ΔChBDs as the starting scaffold, folding free energy calculations identified a stabilizing mutation, A347P, within the CID hydrophobic core, which significantly enhanced thermostability by extending the half-life at 45 °C by 56 min. Based on this stabilized scaffold, evolutionary coupling analysis and stability predictions guided the design of secondary mutations, yielding several double mutants with improved catalytic performance. The best variants (A347P/A137S, A347P/A158G, and A347P/E219A) exhibited increases in specific activity of up to 99% compared with the wild-type and substantially prolonged half-lives at 50 °C. Notably, A347P/A137S produced 4.9-fold more GlcNAc during colloidal chitin hydrolysis. These results demonstrate that CID-targeted engineering provides an effective strategy for optimizing multidomain chitinases.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2026
·Vol. 74
·期 33
·26610-26624
·
IF 6.7
Liang Bicheng,Zhang Chen,Li Guantian,Yang Haoyue,Li Kecheng,Li Rongfeng,Liu Song,Yu Huahua,Xing Ronge
A previously uncharacterized GH18 Chitinase from Bacillus haynesii (BhGH18) was heterologously expressed
and characterized. BhGH18 displayed maximal activity at pH 7.5 and
40 ° C, and activity was enhanced by Mg 2+ /Zn 2+ but inhibited by Cu 2+ /Fe 2+ /Ni 2+ . After further purification, the enzyme exhibited markedly improved
kinetics toward pNP-(GlcNAc) 2 ( K m = 0.103 mM, V max = 0.217 μmol/min).
Importantly, on the natural substrate colloidal chitin, BhGH18 gave
an apparent K m of 3.51 mg/mL and V max of 1.69 μmol/min (for colloidal chitin).
The enzyme displayed an endohydrolytic preference, converting ultrasonically
pretreated chitin predominantly into chitobiose (DP2). Ultrasonication
at 500 W increased the hydrolysis yield nearly 6-fold (23.86% conversion).
Molecular docking identified Trp157, Glu197, Tyr198 and Arg333 as
key substrate-binding residues. This work demonstrates an integrated
ultrasound-enzyme strategy using BhGH18 for sustainable production
of DP2-enriched chitin oligosaccharides.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Shiqing Zhang,Yifan Lin,Zhanhua Zhang,Xingyan Ou,Zhenbo Xu,Chengjuan Zou,Thanapop Soteyome,Lili Li,Xiaomei Lin,Yanrui Ye
Chitin, the second most abundant natural polysaccharide, is a renewable resource with great potential for producing functional oligosaccharides. In this study, we identified and characterized CtChi70, a glycoside hydrolase family 18 (GH18) exochitinase that selectively hydrolyzes chitin to produce N -acetyl chitobiose [(GlcNAc)₂] with exceptional purity (≥98 %). CtChi70 was heterologously expressed in Escherichia coli CtChi70 at a high yield of 228.2 ± 47.8 mg L −1 . The purified enzyme exhibited optimal activity at pH 5.0 and 45 °C, and demonstrated stability across a broad pH range (4.0–12.0). Time-course hydrolysis of colloidal chitin showed moderate catalytic efficiency but high product specificity, peaking at 24 h with minimal N -acetylglucosamine byproduct. The ability of CtChi70 to directly convert raw chitin powder to high-purity (GlcNAc)₂ suggests its potential for applications requiring pure chitooligosaccharides. These findings highlight CtChi70 as a promising biocatalyst for sustainable chitin valorization.
研究领域:
酶学
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分子生物学
·
生物催化
·
碳水化合物化学
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微生物代谢
·
蛋白质工程
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Food Bioscience
·
2025
·
IF 5.9
Chenxi Gu,Liyan Yang,Susu Han,Dengfeng Yang,Xiaodong Xu,WenChao Zhang,Mingguo Jiang,Lixia Pan
Cold-active chitinases that maintain robust catalytic performance at low temperatures are gaining attention as eco-friendly biocatalysts for energy-saving bioprocesses. In this study, a novel multifunctional chitinase of GH18 ( Cm Chi18B) was successfully cloned from a newly identified mangrove-associated bacterium Chitinibacter mangrovi FCG-7 T in Escherichia coli . The enzyme Cm Chi18B demonstrated adaptability to cold environments, retaining 55% of its maximal activity at 0°C. It displayed optimal activity at 55°C and pH 5.5. Cm Chi18B demonstrated high activity across a broad pH range of 4.0-12.0. Substrate hydrolysis analysis indicated that Cm Chi18B possesses bi-functional capabilities, including exochitinase and N-acetyl-β-D-glucosaminidase activities. After 24 h of incubation with colloidal chitin at 30°C, the enzyme achieved a N-acetyl-D-glucosamine (GlcNAc) conversion rate of 91.15% with a purity exceeding 98%. Intriguingly, the hydrolysis time for GlcNAc production varied with temperatures, with 0°C significantly impairing N-acetyl-β-glucosaminidase activity. These attributes position Cm Chi18B as a promising, low-energy biocatalyst for the sustainable production of GlcNAc and the valorization of chitin waste streams.
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Microorganisms
·
2025
·Vol. 13
·期 9
·2033
·
IF 4.2
Yanmei Cai,Xinyu Li,Shuang Chen,Qichao Liu,Hongxiang Lu,Jiahui Xie,Wei Li,Guiying Chen
Microorganisms with chitin-degrading capabilities play a crucial role in the biological control of crop pests and diseases as well as in the treatment of organic waste. In this study, a chitin-degrading bacterium, designated L2-2, was isolated from the intestine ofOdorrana margaretaecollected in Mount Emei, Sichuan, China. Based on physiological and biochemical characteristics, 16S rRNA gene sequencing, and phylogenetic analysis of 31 conserved housekeeping genes in the whole genome, strain L2-2 was identified as a member of the genusRoseateles, namedRoseatelessp. L2-2. This strain is able to grow on agar medium with colloidal chitin as the sole carbon source and form clear hydrolysis zones. After optimizing fermentation conditions (including concentrations of nitrogen and carbon sources, culture time, and pH), the enzyme activity was increased to 3.46 U/mL, which was 24 times higher than the initial enzyme activity. Functional genome annotation showed that the strain contains genes encoding endochitinases of the GH18, GH23, and GH46 families, as well as genes encoding β-glucosidases of the GH1, GH2, GH3, and GH109 families, indicating its genetic basis for chitin-degrading potential. This study expands the diversity of known chitin-degrading bacteria and provides a promising microbial resource for the bioremediation of chitinous waste and sustainable pest control in agriculture.
研究领域:
微生物学
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环境生物技术
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分子生物学
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应用微生物学
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生物修复
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农业微生物学
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Alei Zhang,Runke Wang,Xi Zhou,Quanzhen Liu,Xin Zhang,Feifei Chen,Kequan Chen
In this work, a GH18 chitinase (Chi1) with highly hydrolytic activity was identified from Chitinibacter sp. GC72 and successfully cloned and expressed in Escherichia coli BL21(DE3). The recombinant Chi1 exhibited specific activities of 362.6 U/mg against colloidal chitin and 56.7 U/mg against raw α-chitin. Analysis of hydrolysis pattern showed that Chi1 is a multifunctional chitinase, which mainly exhibited processive exo-acting activity with some endo-acting and N -acetyl glucosaminidase activities. Structural analysis and molecular docking were conducted to explore the potential mechanism underlying the highly and multiple activities. Furthermore, Chi1 was utilized to convert raw α-chitin into reducing sugar affording 48.6% ( w / w ) yield of total sugar with a 1:4 molar ratio of NAG to NAG 2 , while also displaying hydrolysis activity to chitinous biomass with 4.1% sugar yield. This study provides a potent chitinase to hydrolyze chitinous resource with unusual product pattern, showing great potential for further industrial applications.
研究领域:
微生物学
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分子生物学
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生物技术
·
酶学
·
生物信息学
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环境生物技术
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工业生物技术
·
结构生物学
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