Production and characterization of a thermostable endo-β-1,3(4)-glucanase for the combined enzymatic degradation of chitin-glucan to produce prebiotic oligosaccharides from mushroom waste

IF 2.8 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of chemical technology and biotechnology Pub Date : 2025-04-12 DOI:10.1002/jctb.7866

Anastasia Klemanska, Ayman Hijazi, Jesper Holck, Francisco Duarte, Kelly Dwyer, Gary Walsh

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引用次数: 0

Abstract

BACKGROUND

Growing global demand for mushrooms means an increased production of solid mushroom waste (SMW), necessitating novel approaches for waste valorization. Waste stalks and misshapen mushrooms are rich in chitin-glucan polymer – a promising source of prebiotics. Tter16 (EC 3.2.1.6) from Thielavia terrestris was cloned, recombinantly expressed in Pichia pastoris, and biochemically characterized. An enzyme cocktail preparation containing Tter16, two other endo-β-1,3(4)-glucanases (Csph16A and Csph16A-ΔC) and one endo-β-1,6-glucanase (Tvir30) (EC 3.2.1.75), was used to degrade the glucan fraction of fungal chitin-glucan with the aim of valorizing bioactive polymers within commercial mushroom waste.

RESULTS

Tter16 was optimally active at pH 5.0, 50 °C, and hyperthermostable at 50 °C with a D-value of 33 days. The Tm of Tter16 was determined to be 67.77 °C and the substrate specificity analysis revealed a preference for mixed-linkage β-glucans. Tter16 was found to possess one of the highest turnover numbers recorded for noncellulolytic fungal glucanases, with a value of 1221 s⁻¹. The enzyme cocktail yielded oligosaccharides including a β-1,6-linked disaccharide and β-1,3-linked oligosaccharides DP 2–5, while minimizing the production of glucose at >90 min of hydrolysis of chitin-glucan. A small-scale chitin-glucan hydrolysis study resulted in a 242.5 mg yield of soluble sugars per g of substrate (24.3% yield) after 90 min of hydrolysis. These oligosaccharides were found to support the growth of the probiotic Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) in vitro.

CONCLUSION

The biochemical properties of Tter16 are well-suited for the intended application of mushroom waste valorization. Enzymatic hydrolysis of chitin-glucan released β-glucooligosaccharides displaying prebiotic potential. To the best of our knowledge, this is the first account of the cooperative enzymatic degradation of chitin-glucan with the aim of liberating prebiotic β-glucooligosaccharides from solid mushroom waste and their subsequent in vitro application testing. © 2025 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

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一种耐热内切酶-β-1,3(4)-葡聚糖酶的制备及表征，用于联合酶降解几丁质-葡聚糖，从蘑菇废物中生产益生元低聚糖

背景：全球蘑菇需求的增长意味着固体蘑菇废物（SMW）的产量增加，需要新的废物增值方法。废秸秆和畸形蘑菇富含几丁质-葡聚糖聚合物-一种有前途的益生元来源。克隆了地鼠Tter16 (EC 3.2.1.6)，在毕赤酵母中重组表达，并进行了生化表征。利用一种含有Tter16、另外两种内切-β-1,3(4)-葡聚糖酶（Csph16A和Csph16A-ΔC）和一种内切-β-1,6-葡聚糖酶（Tvir30）（EC 3.2.1.75）的鸡尾酒酶制剂，降解真菌几丁质葡聚糖的葡聚糖部分，目的是在商业蘑菇废弃物中产生生物活性聚合物。结果Tter16在pH 5.0、50℃条件下具有最佳活性，在50℃条件下具有超热稳定性，d值为33天。确定Tter16的Tm为67.77°C，底物特异性分析显示对混合连锁β-葡聚糖有偏好。Tter16被发现具有最高的营业额记录的非纤维素水解真菌葡聚糖酶之一，其值为1221 s−1。混合酶产生的低聚糖包括β-1,6连接的双糖和β-1,3连接的低聚糖DP - 2-5，同时在几丁质-葡聚糖水解90分钟时，葡萄糖的产量降至最低。一项小规模的几丁质-葡聚糖水解研究表明，水解90分钟后，每克底物的可溶性糖产量为242.5 mg（收率为24.3%）。这些低聚糖被发现支持益生菌鼠李糖乳杆菌（原鼠李糖乳杆菌）的体外生长。结论Tter16具有良好的生化性能，适合于菌菇废弃物增值的预期应用。酶解几丁质-葡聚糖释放出具有益生元潜力的β-低聚糖。据我们所知，这是第一个关于甲壳素-葡聚糖的协同酶降解，目的是从固体蘑菇废物中释放益生元β-低聚葡萄糖及其随后的体外应用测试。©2025作者。化学技术与生物技术杂志，John Wiley &出版；代表化学工业学会（SCI）的儿子有限公司。

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来源期刊

Journal of chemical technology and biotechnology 工程技术-工程：化工

CiteScore

7.00

自引率

5.90%

发文量

268

审稿时长

1.7 months

期刊介绍： Journal of Chemical Technology and Biotechnology(JCTB) is an international, inter-disciplinary peer-reviewed journal concerned with the application of scientific discoveries and advancements in chemical and biological technology that aim towards economically and environmentally sustainable industrial processes.