
干酪乳杆菌(Lactobacillus casei)HDS-01甘露聚糖酶基因异源表达及功能验证
Mannanase Gene in Lactobacillus casei HDS-01: Heterologous Expression and Functional Verification
为实现甘露聚糖酶基因异源表达并验证功能,本研究以产甘露聚糖酶的副干酪乳杆菌(Lactobacillus casei) HDS-01为供试菌株,将基因组序列中功能预测为甘露糖苷酶的基因M1在大肠杆菌中表达,分别以pET-28a和Escherichia coli BL21(DE3)为载体和宿主菌构建M1基因工程菌株,纯化重组蛋白并验证甘露聚糖降解功能。结果表明:重组蛋白M1在表达过程中以包涵体形式存在,经Ni柱亲和层析纯化后,SDS-PAGE显示蛋白为98 kDa,复性后酶活和蛋白浓度分别为19.24±0.55 U/mL和0.51±0.01 mg/mL。高效液相色谱(High pressure liquid chromatography, HPLC)检测重组蛋白M1降解魔芋粉的产物,反应前3 h,甘露三糖或四糖浓度显著高,此后,甘露糖和甘露二糖浓度逐渐升高。本研究为产甘露聚糖酶乳酸菌直接用于食品级领域奠定了基础,也为乳酸菌甘露聚糖酶蛋白的定向改造提供了依据。
In order to realize heterologous expression of mannanase and verify its functions, the mannanase-producing Lactobacillus casei HDS-01 was used as the test strain in this study. The M1 predicted to be mannosidase was expressed in Escherichia coli. M1 gene engineering strains were constructed using pET-28a as the vector and E. coli BL21 (DE3) as the host. The recombinant protein was purified and the degradation function of mannan was verified. The results indicated that the recombinant protein M1 existed as inclusion body during expression. After purification by Ni affinity chromatography, the size of the protein was 98 kDa. After renaturation, mannanase activity and protein concentration were 19.24±0.55 U/mL and 0.51±0.01 mg/mL, respectively. High pressure liquid chromatography (HPLC) was used to detect the degradation product of Konjac powder. The concentration of mannotriose or tetrasaccharide was significantly higher in the first 3 hours, and mannose and mannobiose gradually increased as the reaction progressing. This study laid the foundation for the direct application of mannanase-producing lactic acid bacteria in food-grade field and provided the basis for the selective modification of mannanase in lactic acid bacteria.
β-甘露聚糖酶 / 干酪乳杆菌 / 异源表达 / 重组蛋白 / 功能验证 {{custom_keyword}} /
β-1,4-mannanase / Lactobacillus casei / heterologous expression / recombinant protein / functional verification {{custom_keyword}} /
[1] |
Diesel fuel injector nozzles have a significant effect on the quality of spray and charge preparation. However, the mechanism and degree of this effect is unclear. The complexity of the internal nozzle flow has hindered the study of the fuel injection process. Diesel fuel injector nozzle flows are highly turbulent and usually two-phase. Several experiments have shown the presence of cavitation in the nozzles to be a dominating effect. Recent experimental work has revealed new qualitative details about the cavitation in fuel injector nozzles. The cavity tends to be smooth near the inlet, transitioning to a more ruffled appearance downstream. These flows are also strongly asymmetric in realistic geometries. Additionally, photographs have shown string cavitation inside the sac volume extending into the nozzles. The strings appear to be a form of stratified two-phase flow, like the cavities near the inlet corner. Like the cavities, the strings break down into bubbly flow near the exit of the nozzle. Future experiments in this field should address the exact nature of the two-phase flow at the exit.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[2] |
陈祺琛, 杨江科, 王兴吉, 等. 耐高温β-甘露聚糖酶基因的高效表达与水解魔芋精粉条件的优化[J]. 食品科技, 2018, 43(8):1-8.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[3] |
In this paper, konjac oligoglucomannan (KOGM) was obtained with a hydrolysis rate of 56.24% by controlling the hydrolysis conditions. KOGM was passed through a 0.2 kDa dialysis bag, a 3 kDa ultrafiltration tube, and a 5 kDa ultrafiltration tube, creating samples with molecular weights of 0.2–3 kDa (IV), 3–5 kDa (III), and >5 kDa (II), respectively. The in vitro antioxidant activities of the KOGM samples were tested by measuring their removal effects on ˙OH, {\text{O}}_{2}^{-}, and DPPH˙. The in vivo antioxidant activities of the samples were analyzed by measuring their impacts on the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-PX) activity in mice. The results show that the KOGM samples in groups III and IV could effectively remove ˙OH, {\text{O}}_{2}^{-}, and DPPH˙; the KOGM samples in all three groups could enhance the SOD and GSH-PX activities and reduce the MDA content in the liver tissues of mice; finally, the antioxidant activity of KOGM is negatively correlated with the molecular weight.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[4] |
仝泽方, 李利军, 卢美欢, 等. 葡甘聚糖酶制备魔芋葡甘露低聚糖的工艺研究[J]. 中国调味品, 2021, 46(7):167-170.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[5] |
A GalNAc/Gal-specific lectins named CGL and MTL were isolated and characterized from the edible mussels Crenomytilus grayanus and Mytilus trossulus. Amino acid sequence analysis of these lectins showed that they, together with another lectin MytiLec-1, formed a novel lectin family, adopting β-trefoil fold. In this mini review we discuss the structure, oligomerization, and carbohydrate-binding properties of a novel lectin family. We describe also the antibacterial, antifungal, and antiproliferative activities of these lectins and report about dependence of activities on molecular properties. Summarizing, CGL, MTL, and MytiLec-1 could be involved in the immunity in mollusks and may become a basis for the elaboration of new diagnostic tools or treatments for a variety of cancers.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[6] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[7] |
殷运菊, 闫昭明, 陈清华, 等. β-甘露聚糖酶的结构,特性及其在畜禽生产中的应用[J]. 动物营养学报, 2021, 33(5):1-9.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[8] |
汤文晶, 李松, 马忠宝, 等. 碱性β-甘露聚糖酶产生菌的筛选,鉴定及发酵条件优化[J]. 食品与发酵工业, 2019, 45(23):50-54.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[9] |
郭尚旭, 王瑶, 那金, 等. 细菌β-甘露聚糖酶研究进展[J]. 中国农学通报, 2017, 33(27):61-65.
甘露聚糖酶是能够水解任意β-1,4-糖苷键的内切水解酶,广泛存在于动植物和微生物中。微生物中的细菌甘露聚糖酶,通常具有更小的分子量,与其他来源的甘露聚糖酶相比,具有成本低、发酵周期短、容易提取,及最适反应pH和温度耐受区间都更加宽泛等优势,广泛应用于饲料,食品,石油开采,造纸等领域中。因此,本文将对细菌甘露聚糖酶的来源、性质、分子生物学及其在饲料生产中的应用等方面进行综述,以期为细菌甘露聚糖酶进一步开发利用提供基础。
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[10] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[11] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[12] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[13] |
张丹阳, 吕育财, 田毅红, 等. 枯草芽孢杆菌β-甘露聚糖酶的克隆表达及重组酶性质研究[J]. 食品工业科技, 2020, 41(6):88-105.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[14] |
黄承敏, 肖茜, 王蓉蓉, 等. 一株高产胞外多糖乳酸菌的分离鉴定及其产胞外多糖的研究[J]. 中国酿造, 2019, 38(1):80-83.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[15] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[16] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[17] |
季海蕊, 张鑫, 姜静, 等. 乳酸菌β-甘露聚糖酶研究进展[J]. 食品工业科技, 2021, 42(4):325-336.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[18] |
杜仁鹏, 赵丹, 王晓宇, 等. 1株乳酸高产菌的分离鉴定与系统发育分析[J]. 中国食品学报, 2017, 17(9):248-255.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[19] |
张鑫, 王瑶, 姜静, 等. 干酪乳杆菌(Lactobacillus casei)HDS-01产甘露聚糖酶条件研究[J]. 中国农学通报, 2020, 36(29):78-86.
旨在提高分离自东北酸菜发酵液中的干酪乳杆菌(Lactobacillus casei)HDS-01产酶水平,为甘露聚糖酶的分离纯化和工业生产奠定基础,以MRS为初始培养基,设置不同培养基组分及发酵条件进行单因素试验,用DNS法和平板菌落计数法分别测定酶活和L. casei HDS-01生长量。试验实验结果表明 L. casei HDS-01产甘露聚糖酶的最优条件如下:最适碳源及浓度为0.8%的果胶,最适氮源及浓度为0.2%的酵母提取物和硝酸铵,无机盐种类及浓度包括0.2%的柠檬酸铵、0.1%的磷酸氢二钾、0.9%的乙酸钠,最适装液量为100 mL/250 mL,最适接种量为7%,最适培养温度为37℃,初始pH 5.5。按照上述最适条件,在摇床转速为140 r/min条件下培养36 h后,酶活可达72.7±0.30 U/mL,较优化前的产酶水平提升了6.51倍。
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[20] |
刘学, 卢海强, 王玉印, 等. 甘露聚糖酶AfMan5A和RmMan134C的性质差异分析及对多糖的协同降解[J]. 食品科学, 2022, 43(24):145-153.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[21] |
马鑫, 赵仲麟, 李亮, 等. 芽孢杆菌β-甘露聚糖酶基因的克隆及表达[J]. 生物技术通报, 2009(2):77-81.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[22] |
朱泾, 赵述淼, 彭楠, 等. 冰岛硫化叶菌β-1,4-内切葡聚糖酶的同源表达、纯化与性质[J]. 华中农业大学学报, 2011, 30(6):674-679.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[23] |
董墨思, 解婉莹, 李苏红. 部分酶解瓜尔豆胶功能性的研究进展[J]. 食品工业, 2016, 37(9):212-215.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[24] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[25] |
者园园, 苗华彪, 吴倩, 等. 耐热β-甘露聚糖酶的异源表达及低聚寡糖制备研究[J]. 饲料研究, 2021(23):63-68.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[26] |
甄红敏, 华晓晗, 马俊文, 等. 产黄青霉β-甘露聚糖酶的高效表达、性质及应用[J]. 食品科学, 2021, 42(8):98-105.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
[27] |
郭金玲, 张丹阳, 樊雪莲, 等. β-甘露聚糖酶产生菌的筛选及魔芋低聚糖制备工艺的研究[J]. 中国酿造, 2018, 37(8):123-127.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
{{custom_ref.label}} |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
/
〈 |
|
〉 |