嗜热小孢根霉GH134家族甘露聚糖酶基因的克隆表达及在果汁加工中的应用

doi:10.7506/spkx1002-6630-20220928-312

食品科学 ›› 2023, Vol. 44 ›› Issue (16): 177-184.doi: 10.7506/spkx1002-6630-20220928-312

嗜热小孢根霉GH134家族甘露聚糖酶基因的克隆表达及在果汁加工中的应用

刘学，韩璞，陈伟，田洪涛，谷新晰

（1.河北农业大学食品科技学院，河北保定 071000；2.河北保定第二中学，河北保定 071000）

出版日期:2023-08-25 发布日期:2023-09-01
基金资助:
国家自然科学基金青年科学基金项目（31901631）；保定市教育科学研究“十四五”规划项目（2202076）

Cloning and Expression of a Novel GH134 β-Mannanase Gene from Thermophilic Fungus Rhizopus microsporus and Its Application in Juice Processing

LIU Xue, HAN Pu, CHEN Wei, TIAN Hongtao, GU Xinxi

(1. College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China;2. NO.2 High School of Baoding, Baoding 071000, China)

Online:2023-08-25 Published:2023-09-01

摘要/Abstract

摘要： 本研究从浓香大曲中筛选出1 株嗜热真菌HBFH10，经鉴定为小孢根霉（Rhizopus microsporus）。利用兼并引物，从菌株HBFH10中获得1 个新型甘露聚糖酶基因（RmMan134），该基因全长552 bp，无内含子，编码183 个氨基酸和1 个终止密码子。RmMan134酶蛋白分子由1 个信号肽序列（1～19 aa）和GH134家族的催化区组成。经异源表达后，重组RmMan134的最适反应温度为50 ℃，最适反应pH值为6.0，具有较强的热稳定性和pH值稳定性。RmMan134成熟蛋白分子质量约为18.5 kDa，存在糖基化修饰。经动力学性质测定，以魔芋胶为底物时，该酶的Vmax为57.1 μmol/（min·mg），Km为0.66 mg/mL。重组酶RmMan134对橙汁的澄清效果最为明显，澄清度提高了23.8%，其次苹果汁提高了11%，桃汁提高了7%左右。除此之外，添加重组酶使得葡萄汁的出汁率提高了7%左右。综上所述，甘露聚糖酶RmMan134在果汁加工中具有较好的应用前景。

关键词: 大曲；嗜热真菌；小孢根霉；甘露聚糖酶；GH134家族；果汁加工

Abstract: A thermophilic fungus, named HBFH10, was isolated from Luzhou-flavor Daqu and identified as Rhizopus microsporus. A new β-mannanase gene (RmMan134) from Rhizopus microsporus HBHF10, was cloned using degenerate primers, expressed, and characterized. The cloned RmMan134 gene had a total length of 552 bp without introns, encoding 183 amino acids and one stop codon. The expressed enzyme was composed of one signal peptide sequence of 19 amino acid residues and the catalytic region of the GH134 family. The optimal pH and temperature for the recombinant enzyme were 6.0 and 50 ℃, respectively. It had high thermal and pH stability. Its molecular mass was estimated to be approximately 18.5 kDa, and it was a glycosylated molecule. When konjac gum was used as the substrate, the Km and Vmax values of RmMan134 were 0.66 mg/mL and 57.1 μmol/(min·mg), respectively. RmMan134 exhibited the best clarification efficiency for orange juice, resulting in a 23.8% increase in its clarity, followed apple juice (11%) and peach juice (7%). Moreover, RmMan134 significantly increased the yield of grape juice by about 7%. Overall, RmMan134 has good application prospects in juice processing.

Key words: Daqu; thermophilic fungus; Rhizopus microsporus; mannanase; GH134 family; juice processing

中图分类号:

Q939.97

刘学，韩璞，陈伟，田洪涛，谷新晰. 嗜热小孢根霉GH134家族甘露聚糖酶基因的克隆表达及在果汁加工中的应用[J]. 食品科学, 2023, 44(16): 177-184.

LIU Xue, HAN Pu, CHEN Wei, TIAN Hongtao, GU Xinxi. Cloning and Expression of a Novel GH134 β-Mannanase Gene from Thermophilic Fungus Rhizopus microsporus and Its Application in Juice Processing[J]. FOOD SCIENCE, 2023, 44(16): 177-184.

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嗜热小孢根霉GH134家族甘露聚糖酶基因的克隆表达及在果汁加工中的应用

Cloning and Expression of a Novel GH134 β-Mannanase Gene from Thermophilic Fungus Rhizopus microsporus and Its Application in Juice Processing

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