Megazyme K-CELLG5-2V 内切纤维素酶检测试剂盒(CELLG5方法)
中文名称：内切纤维素酶检测试剂盒(CELLG5方法)
英文名：Cellulase Assay Kit (CELLG5 Method)
货号：K-CELLG5-2V
规格：60 / 120 assays (manual) / 240 (auto-analyser)
The CellG3 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components;
1) 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-D-cellotrioside (BCNPG3) and 2) Thermostable β-glucosidase. The benzylidene blocking group prevents any hydrolytic action by the β-glucosidase on BCNPG3. Incubation with an endo-cellulase generates a non-blocked colourimetric oligosaccharide that is rapidly hydrolysed by the ancillary β-glucosidase. The rate of formation of 2-chloro-4-nitrophenol is therefore directly related to the hydrolysis of BCNPG3 by the endo-cellulase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH 9.0).
用于测量内切纤维素酶（内切1,4-β-葡聚糖酶）的CellG3分析试剂包含两个成分;
1）4,6-O-亚苄基-2-氯-4-硝基苯基-β-D-纤维二糖苷（BCNPG3）和2）恒温β-葡萄糖苷酶。 亚苄基保护基团可防止β-葡萄糖苷酶对BCNPG3的任何水解作用。 与内切纤维素酶一起孵育会生成不封闭的比色寡糖，该寡糖会被辅助β-葡萄糖苷酶快速水解。 因此，2-氯-4-硝基苯酚的形成速率与内纤维素酶水解BCNPG3直接相关。 加入Tris缓冲溶液（pH 9.0）后，反应终止，苯酚盐显色。
Please note that a new assay kit (K-CellG5) is now available for the measurement of endo-cellulase. The CellG5 reagent contains a cellopentaose core and exhibits vastly improved sensitivity for some cellulases. In addition, the exchange of the benzylidene blocking group in CellG3 for 3-keto-butylidene in CellG5 improves the substrate’s water solubility significantly, allowing for a reduction in the concentration of DMSO required in the assay. As DMSO is known to inhibit certain cellulases, this is another benefit in using CellG5. Megazyme now recommends the use of K-CellG5 for all assays for the measurement of endo-cellulase.
请注意，现在可以使用新的测定试剂盒（K-CellG5）测量内切纤维素酶。 CellG5试剂含有一个纤维戊糖核心，对某些纤维素酶的敏感性大大提高。 此外，CellG3中的亚苄基保护基团换成CellG5中的3-酮丁烯基可以显着提高底物的水溶性，从而降低了测定所需的DMSO浓度。 由于已知DMSO会抑制某些纤维素酶，因此这是使用CellG5的另一个好处。 Megazyme现在建议在所有测定内切纤维素酶的测定中使用K-CellG5。
Suitable for auto/analyser formats.
Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase).
McCleary, B. V., Mangan, D., Daly, R., Fort, S., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 385, 9-17.
Read Abstract
A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of Thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and reADIly adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.
制备了用于检测内切-1,4-β-葡聚糖酶（纤维素酶）的特异性和灵敏底物。 在可加热的β-葡萄糖苷酶存在下，底物混合物包含亚苄基末端封闭的2-氯-4-硝基苯基-β-纤维二糖苷（BzCNPG3）。 非还原性末端D-葡萄糖基残基上亚苄基的存在可防止β-葡糖苷酶和exo-β-葡聚糖酶等exo-acting酶的水解。 通过纤维素酶水解后，2-氯-4-硝基苯基-β-糖苷立即被底物混合物中的β-葡萄糖苷酶水解为2-氯-4-硝基苯酚和游离的D-葡萄糖。 通过加入弱碱性溶液终止反应并显色。 该测定方法易于使用，特异性，准确，鲁棒并且非常适合于自动化。 一般而言，该程序应在生物质酶学和内切-1,4-β-葡聚糖酶的特异性测定中找到广泛的应用。
Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase.
Mangan, D., McCleary, B. V., Liadova, A., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 395, 47-51.
Read Abstract
There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.
A novel automatable enzyme-coupled colorimetric assay for endo-1,4-β-glucanase (cellulase).
Mangan, D., Cornaggia, C., McKie, V., Kargelis. T. & V. McCleary, B. V. (2016). Analytical and Bioanalytical Chemistry, 408(15), 4159-4168.
Read Abstract
endo-1,4-β-Glucanase (endo-cellulase, EC 3.2.1.4) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside.
Colourimetric method for the determination of endo-1,4-β-
glucanase (cellulase) in enzyme preparations and fermentation
products
Colourimetric method for the determination of
endo-1,4-β-glucanase (cellulase) in enzyme preparations and fermentation products
Principle:
(endo-1,4-β-glucanase)
(1) 3-Ketobutylidene-G5-β-PNP + H2O → Blocked-GX + G(5-X)-β-PNP
(thermostable β-glucosidase)
(2) G(5-X)-β-PNP + H2O → D-glucose + PNP
(alkaline solution)
(3) PNP → phenolate ion (yellow colour)
Note: PNP = 4-nitrophenol
Kit size:
K-CELLG5-4V 120 / 240 assays (manual) / 480 (auto-analyser)
or
K-CELLG5-2V 60 / 120 assays (manual) / 240 (auto-analyser)
Method: Spectrophotometric at 400 nm
Total assay time: 10 min
Detection limit: 3.5 x 10-4 U/mL
Application examples:
Fermentation broths, industrial enzyme preparations, biofuels research
Method recognition: Novel method
Advantages
- Very cost effective
- All reagents stable for > 4 years
- Completely specific for cellulase (endo-1,4-glucanase)
- Generally applicable and highly sensitive
- Simple format. Well suited to automation
- Standard included
| Content: | (K-CellG5-2V) 60 / 120 assays (manual) / 240 assays (auto-analyser) or (K-CellG5-4V) 120 / 240 assays (manual) / 480 assays (auto-analyser) |
| Shipping Temperature: | Ambient |
| Storage Temperature: | Short term stability: 2-8oC, Long term stability: See individual component labels |
| Stability: | > 2 years under recommended storage conditions |
| Analyte: | endo-Cellulase |
| Assay Format: | Spectrophotometer, Auto-analyser |
| Detection Method: | Absorbance |
| Wavelength (nm): | 400 |
| Signal Response: | Increase |
| Limit of Detection: | 1.2 x 10-3 U/mL |
| Reproducibility (%): | ~ 3% |
| Total Assay Time: | 10 min |
| Application examples: | Fermentation broths, industrial enzyme preparations and biofuels research. |
| Method recognition: | Novel method |
Cellulase Activity Assay Kit.
The CellG5 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components;
1) 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside (BPNPG5) and 2) thermostable β-glucosidase. The ketone blocking group prevents any hydrolytic action by the β-glucosidase on BPNPG5. Incubation with an endo-cellulase generates a non-blocked colourimetric oligosaccharide that is rapidly hydrolysed by the ancillary β-glucosidase. The rate of formation of 4-nitrophenol is therefore directly related to the hydrolysis of BPNPG5 by the endo-cellulase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH 9.0).
The CellG5 assay represents a huge step forward in the methodology for the measurement of cellulase that traditionally relied on substrates such as CM-cellulose, Avicel, cellooligosaccharides, filter paper or dyed polysaccharides including CMC Congo red or cellulose azure.
| 内容： | (K-CellG5-2V) 60/120检测(手册)/240(自动分析仪)或 (K-CellG5-4V) 120/240检测(手册)/480(自动分析仪) |
| 船运温度： | 环境 |
| 储存温度： | 短期稳定性：2-8oC， 长期稳定性：见单个组件标签 |
| 稳定性： | >2年在建议的储存条件下 |
| 分析物： | 恩藤-纤维素酶 |
| 化验格式： | 分光光度计，自动分析仪 |
| 检测方法： | 吸光度 |
| 波长(纳米)： | 400 |
| 信号响应： | 增加 |
| 检测极限： | 1.2 x 10-3U/mL |
| 可重复性(%)： | ~ 3% |
| 试验总时间： | 10分钟 |
| 应用实例： | 发酵液、工业酶制剂和生物燃料研究。 |
| 方法识别： | 新方法 |
纤维素酶活性分析试剂盒。
CellG 5测定试剂恩藤-纤维素酶(恩藤-1，4-β-葡聚糖酶含有两个组分；
1) 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside(BPNPG 5)和2)耐热β-葡萄糖苷酶。酮阻断组阻止β-葡萄糖苷酶对BPNPG 5的任何水解作用.与恩藤-纤维素酶产生一种非封闭的色测低聚糖，由辅助的β-葡萄糖苷酶迅速水解.因此，4-硝基酚的生成速度直接关系到BPNPG 5的水解。恩藤-纤维素酶在pH为9.0的Tris缓冲溶液中，反应终止，显色。
在纤维素酶的测定方法上，CellG 5是向前迈进了一大步。纤维素酶传统上依赖于底物，如CM-纤维素、Avicel、纤维低聚糖、滤纸或染色多糖，包括CMC、刚果红或纤维素天蓝。