CN101967470B - Cellulase stabilizing auxiliary agent composition - Google Patents
Cellulase stabilizing auxiliary agent composition Download PDFInfo
- Publication number
- CN101967470B CN101967470B CN201010537963A CN201010537963A CN101967470B CN 101967470 B CN101967470 B CN 101967470B CN 201010537963 A CN201010537963 A CN 201010537963A CN 201010537963 A CN201010537963 A CN 201010537963A CN 101967470 B CN101967470 B CN 101967470B
- Authority
- CN
- China
- Prior art keywords
- cellulase
- component
- stabilization aid
- neutral
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 41
- 229940106157 cellulase Drugs 0.000 title claims abstract description 40
- 239000000203 mixture Substances 0.000 title claims abstract description 22
- 230000000087 stabilizing effect Effects 0.000 title abstract description 8
- 239000012752 auxiliary agent Substances 0.000 title abstract 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical group [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000007935 neutral effect Effects 0.000 claims abstract description 11
- GUBGYTABKSRVRQ-QKKXKWKRSA-N lactose group Chemical group OC1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 10
- 108010010803 Gelatin Proteins 0.000 claims abstract description 8
- 239000008273 gelatin Substances 0.000 claims abstract description 8
- 229920000159 gelatin Polymers 0.000 claims abstract description 8
- 235000019322 gelatine Nutrition 0.000 claims abstract description 8
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 8
- 239000008101 lactose Substances 0.000 claims abstract description 8
- 150000001720 carbohydrates Chemical class 0.000 claims abstract 4
- 238000000034 method Methods 0.000 claims description 11
- 230000006641 stabilisation Effects 0.000 claims description 11
- 238000011105 stabilization Methods 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 3
- 241000235058 Komagataella pastoris Species 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- 230000008521 reorganization Effects 0.000 claims 3
- 239000013543 active substance Substances 0.000 claims 2
- 230000008719 thickening Effects 0.000 claims 2
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000005728 strengthening Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 16
- 239000004094 surface-active agent Substances 0.000 abstract description 6
- 239000003755 preservative agent Substances 0.000 abstract description 5
- 239000002562 thickening agent Substances 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000002335 preservative effect Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract 1
- 238000005215 recombination Methods 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 27
- 102000004190 Enzymes Human genes 0.000 description 27
- 229940088598 enzyme Drugs 0.000 description 23
- 239000000243 solution Substances 0.000 description 10
- 238000003860 storage Methods 0.000 description 8
- 108010084185 Cellulases Proteins 0.000 description 5
- 239000000654 additive Substances 0.000 description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
- 102000005575 Cellulases Human genes 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 150000002191 fatty alcohols Chemical class 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 229920000056 polyoxyethylene ether Polymers 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 240000006794 Volvariella volvacea Species 0.000 description 1
- 235000004501 Volvariella volvacea Nutrition 0.000 description 1
- JDZJVWAHZYIHFA-UHFFFAOYSA-N [Br].C1(=CC=CC=C1)O Chemical compound [Br].C1(=CC=CC=C1)O JDZJVWAHZYIHFA-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229940051841 polyoxyethylene ether Drugs 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- SPOMEWBVWWDQBC-UHFFFAOYSA-K tripotassium;dihydrogen phosphate;hydrogen phosphate Chemical compound [K+].[K+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O SPOMEWBVWWDQBC-UHFFFAOYSA-K 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
技术领域 technical field
本发明涉及一种纤维素酶，具体涉及一种纤维素酶稳定助剂组合物。The invention relates to a cellulase, in particular to a cellulase stabilization assistant composition.
背景技术 Background technique
内切型纤维素酶(Endoglucanase)是纤维素酶系中重要组分，在纤维素水解中起着重要作用，它和纤维二糖水解酶(Cellobihydrolase)和β-葡萄糖苷酶(β1，4-Glucosidase)一起协同作用将纤维素水解成葡萄糖，因此，内切型纤维素酶在木质纤维生物炼制，食品工业，纺织工业及造纸工业中均有广泛的应用。从我国草菇(Volvariella volvacea)中克隆到一种新的中性内切型纤维素酶的基因(egI)(Genbank No.AF329732)，与一般的真菌纤维素酶不同，该酶在中性pH环境中具有最高活性和较强的热稳定性，有望成为理想的纺织及造纸工业用纤维素酶。Endo-cellulase (Endoglucanase) is an important component of cellulase system and plays an important role in the hydrolysis of cellulose. Glucosidase) act synergistically to hydrolyze cellulose into glucose. Therefore, endo-cellulase is widely used in lignocellulosic biorefining, food industry, textile industry and paper industry. A new neutral endo-cellulase gene (egI) (Genbank No.AF329732) was cloned from Volvariella volvacea in my country. Unlike common fungal cellulase, the enzyme operates at neutral pH It has the highest activity and strong thermal stability in the environment, and it is expected to become an ideal cellulase for textile and paper industry.
在酶制剂工业化应用过程中，酶不可避免需要在一定条件进行长时间的运输和储存这样一个流通环节。作为一种生物催化剂，特别是在常温条件下，液体酶的活性在长时间的运输和储存过程中极易受到环境因素变化以及微生物污染的影响。细小的环境因素如温度，压力，pH及离子强度的变化可能引起使用酶蛋白的变性失活，同时对微生物来说，酶液也是一种十分丰富的营养液，在常温条件下极易导致微生物的生长，引起酶蛋白的生物降解失活。因此，如何尽可能地保持酶在运输、储存过程中的稳定性是任何一种酶制剂能否最终在相关领域中获得工业化应用的首要前提。In the process of industrial application of enzyme preparations, enzymes inevitably need to be transported and stored under certain conditions for a long period of time. As a biocatalyst, especially at room temperature, the activity of liquid enzymes is easily affected by changes in environmental factors and microbial contamination during long-term transportation and storage. Minor environmental factors such as changes in temperature, pressure, pH and ionic strength may cause the denaturation and inactivation of the enzyme protein used. At the same time, for microorganisms, the enzyme solution is also a very rich nutrient solution, which can easily lead to microorganisms under normal temperature conditions. growth, resulting in inactivation of enzyme protein biodegradation. Therefore, how to maintain the stability of the enzyme during transportation and storage as much as possible is the first prerequisite for any kind of enzyme preparation to finally obtain industrial application in related fields.
提高酶的稳定性，目前常用的方法有：(1)自然筛选或蛋白质工程手段获的高稳定性的酶蛋白；(2)化学修饰和固定化处理；(3)添加保护剂。添加保护剂具有方法简单、成本低下和易于操作等多方面原因，是目前工业上提高酶稳定性的最重要手段之一。从目前国内外研究报道看，添加盐类，糖类，多元醇，以及其它无机有机化合物对提高酶的稳定性都有非常理想的效果，但是添加剂的选择取决于酶的自身特性，这些研究还处于起始阶段，距离工业化生产尚有一段距离。The commonly used methods to improve the stability of enzymes are: (1) highly stable enzyme proteins obtained by natural screening or protein engineering; (2) chemical modification and immobilization; (3) adding protective agents. Adding a protective agent has many reasons, such as simple method, low cost and easy operation, and is one of the most important means to improve enzyme stability in industry at present. From the current research reports at home and abroad, adding salts, sugars, polyols, and other inorganic organic compounds have very ideal effects on improving the stability of enzymes, but the choice of additives depends on the characteristics of the enzyme itself. In the initial stage, there is still a distance from industrialized production.
发明内容 Contents of the invention
发明目的：针对现有技术中存在的不足，本发明的目的是提供一种纤维素酶稳定助剂组合物，以使添加了该添加剂的纤维素酶其具长时间的运输和存储后仍具有较好的酶活力。Purpose of the invention: for the deficiencies in the prior art, the purpose of the present invention is to provide a cellulase stabilizing aid composition, so that the cellulase added with the additive still has a long-term transportation and storage. better enzyme activity.
技术方案：为了实现上述发明目的，本发明采用的技术方案如下：一种纤维素酶稳定助剂组合物，包括以下各组分：2～4g/100mL增稠剂、0.05～0.15mL/100mL表面活性剂、4～6g/100mL糖类和0.2～0.4g/100mL防腐剂；其中，糖类为葡萄糖、蔗糖或乳糖，表面活性剂为AEO系列的表面活性剂，增稠剂为明胶或山梨醇，防腐剂为山梨酸钾、苯甲酸钠或叠氮化钠。AEO是脂肪醇聚氧乙烯醚的缩写，是醇醚，是非离子型表面活性剂中发展最快、用量最大的品种，亲油基和亲水基分别是由具有活泼氢的脂肪醇和环氧乙烷聚合制得，是环氧乙烷加成数不同的多种聚氧乙烯醚的混合物。AEO4是这类产品中的一种。Technical solution: In order to achieve the purpose of the above invention, the technical solution adopted in the present invention is as follows: a cellulase stabilization aid composition comprising the following components: 2-4g/100mL thickener, 0.05-0.15mL/100mL surface Active agent, 4-6g/100mL sugar and 0.2-0.4g/100mL preservative; among them, sugar is glucose, sucrose or lactose, surfactant is AEO series surfactant, thickener is gelatin or sorbitol , preservatives are potassium sorbate, sodium benzoate or sodium azide. AEO is the abbreviation of fatty alcohol polyoxyethylene ether. It is an alcohol ether. It is the fastest growing and most used variety of nonionic surfactants. The lipophilic group and the hydrophilic group are respectively composed of fatty alcohol and ethylene oxide with active hydrogen. It is prepared by the polymerization of alkanes, and it is a mixture of various polyoxyethylene ethers with different addition numbers of ethylene oxide. AEO 4 is one such product.
所述的糖类，优选为乳糖。The sugar is preferably lactose.
所述的增稠剂，优选为明胶。The thickener is preferably gelatin.
所述的表面活性剂，优选为AEO4。The said surfactant is preferably AEO 4 .
所述的防腐剂，优选为叠氮化钠。The preservative is preferably sodium azide.
所述的纤维素酶稳定助剂组合物，优选包括以下各组分：4g/100mL明胶、0.1mL/100mL AEO4、4g/100mL乳糖和0.4g/100mL叠氮化钠。The cellulase stabilization aid composition preferably includes the following components: 4g/100mL gelatin, 0.1mL/100mL AEO 4 , 4g/100mL lactose and 0.4g/100mL sodium azide.
制备上述的纤维素酶稳定助剂组合物的方法，按各组份的添加浓度取各组分，充分混合制得纤维素酶稳定助剂组合物。The method for preparing the above-mentioned cellulase stabilizing aid composition is to take each component according to the added concentration of each component, and fully mix to prepare the cellulase stabilizing aid composition.
上述的纤维素酶稳定助剂组合物在用于增强重组中性纤维素酶稳定性中的应用。Application of the above-mentioned cellulase stabilization aid composition for enhancing the stability of recombinant neutral cellulase.
有益效果：本发明的纤维素酶稳定助剂组合物，组分简单，制备方便，在重组中性纤维素酶稳定性中应用，25℃下储存60d后，最高可使纤维素酶活保留率由47.5％提高到95％，且能较好的抑制微生物的生长。Beneficial effects: the cellulase stabilization aid composition of the present invention has simple components and is easy to prepare. It is used in the stability of recombinant neutral cellulase. After being stored at 25°C for 60 days, the highest retention rate of cellulase activity can be achieved. Increased from 47.5% to 95%, and can better inhibit the growth of microorganisms.
附图说明 Description of drawings
图1是具体实施方式中各实施例具体参数和试验结果表。Fig. 1 is a table of specific parameters and test results of each embodiment in the specific embodiment.
图2是纤维素酶液的SDS-PAGE电泳图。其中，1泳道为原始纤维素酶液，2泳道为25℃，添加添加剂保存60天后的纤维素酶液。Fig. 2 is the SDS-PAGE electrophoresis picture of the cellulase solution. Among them,
具体实施方式 Detailed ways
下面结合附图和具体实施例对本发明做进一步的解释。The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments.
以下实施例所使用的材料如下：The materials used in the following examples are as follows:
重组中性内切型纤维素酶由工程菌P.pastoris菌株S115MEG1Mut+高密度发酵获得，在4℃条件下，5000r/min离心10min后，-80℃条件下保存备用。CMC-Na，美国Sigma公司；其他所用试剂均为国产分析纯试剂。Recombinant neutral endo-cellulase is obtained by high-density fermentation of engineering bacteria P. pastoris strain S115MEG1Mut + , centrifuged at 5000r/min for 10min at 4°C, and stored at -80°C for future use. CMC-Na, Sigma Company, USA; other reagents used were domestic analytical reagents.
实施例1～9Examples 1-9
一种纤维素酶稳定助剂组合物，包括以下各组分：明胶、AEO4、乳糖和叠氮化钠；按各组份的添加浓度取各组分，充分混合制得纤维素酶稳定助剂组合物。A cellulase stabilizing aid composition, comprising the following components: gelatin, AEO 4 , lactose and sodium azide; each component is taken according to the added concentration of each component, and fully mixed to obtain a cellulase stabilizing aid agent composition.
上述的纤维素酶稳定助剂组合物在用于增强重组中性纤维素酶稳定性中的应用。将添加剂组合物加入重组中性纤维素酶，混合均匀，在40℃下保温63h加速试验后，测定残余酶活性，进一步在25℃条件下进行长时间储存验证，60d后测定残余酶活性。各实施例具体参数和试验结果见图1。图1中，R称为极差，数值大反映该因素的影响较大，助剂添加量对酶稳定性的影响顺序为：叠氮化钠＞明胶＞AE04＞乳糖。此表数据为40℃下保温63h的数据，最适配方条件下做了验证(25℃，60d)。酶活保留率＝储存后酶活性/储存前酶活性×100％。Application of the above-mentioned cellulase stabilization aid composition for enhancing the stability of recombinant neutral cellulase. The additive composition was added to the recombinant neutral cellulase, mixed evenly, and incubated at 40°C for 63 hours. After the accelerated test, the residual enzyme activity was measured, and further stored at 25°C for long-term storage verification, and the residual enzyme activity was measured after 60 days. The specific parameters and test results of each embodiment are shown in Figure 1. In Figure 1, R is referred to as extreme difference, and a large value reflects a greater influence of this factor, and the order of the influence of the additive amount on the enzyme stability is: sodium azide > gelatin > AE0 4 > lactose. The data in this table is the data of 63h at 40°C, and it was verified under the most suitable formula conditions (25°C, 60d). Enzyme activity retention rate = enzyme activity after storage/enzyme activity before storage × 100%.
经检测，60d后，酶活保留率为95％，细菌和真菌的数量分别为1.4×109和3.7×108，经SDS-PAGE检测，60d后的目的蛋白条带与保存前没有太大区别，如图2所示，说明60d后酶蛋白降解很少。After testing, after 60 days, the enzyme activity retention rate was 95%, and the numbers of bacteria and fungi were 1.4×10 9 and 3.7×10 8 respectively. After SDS-PAGE detection, the band of the target protein after 60 days was not too large compared with before storage The difference, as shown in Figure 2, shows that there is little enzyme protein degradation after 60 days.
实施例10Example 10
内切型纤维素酶活是以CMC-Na为底物，用Somogyi-Nelson方法进行测定，具体过程按Ding et al方法进行，在1.5mL总反应体系中含有1.0mLpH7.5的KH2PO4-K2HPO4缓冲溶液、0.4mL2％CMC和0.1mL经适当后稀释纤维素酶液，50℃水浴反应30min，加入0.5mLSomogyi试剂煮沸15min，冷却到室温，加入0.5mLNelson试剂，静置20min，10000r/min离心10min后，520nm下测上清液的吸光值，酶活定义为每分钟产生1μmol葡萄糖的酶量为1单位(IU)的酶活。(空白为0.1mL的蒸馏水替换0.1mL纤维素酶的反应样)。The endo-cellulase activity is determined by the Somogyi-Nelson method with CMC-Na as the substrate. The specific process is carried out according to the method of Ding et al. The 1.5mL total reaction system contains 1.0mL of pH7.5 KH 2 PO 4 - K 2 HPO 4 buffer solution, 0.4
SDS-PAGE按Laemmli方法进行，一定量的储存前后得酶液用样品缓冲液(0.06MTris；25％(v/v)甘油；2％(w/v)SDS；0.1％(w/v)溴酚蓝；1％(v/v)β-巯基乙醇)于100℃处理10min变性，浓缩胶浓度3.75％，分离胶浓度7.5％。考马斯亮蓝R250染色。SDS-PAGE was carried out according to the Laemmli method, and a certain amount of enzyme solution was obtained before and after storage with sample buffer (0.06M Tris; 25% (v/v) glycerol; 2% (w/v) SDS; 0.1% (w/v) bromine Phenol blue; 1% (v/v) β-mercaptoethanol) was denatured at 100°C for 10 minutes, the concentration of the stacking gel was 3.75%, and the concentration of the separating gel was 7.5%. Coomassie brilliant blue R250 staining.
细菌和真菌的数量的测定方法：将保存0d、30d、60d的纤维素酶与无菌水按100倍、200倍、500倍、1000倍、2000倍的梯度稀释，取0.1mL的稀释液涂LB板37℃下培养细菌，涂PDA板28℃下培养真菌，隔夜培养后观察菌落并计数。The method of measuring the number of bacteria and fungi: Dilute the cellulase stored for 0d, 30d, and 60d with sterile water in a gradient of 100 times, 200 times, 500 times, 1000 times, and 2000 times, and take 0.1mL of the diluted solution to apply Bacteria were cultured on LB plates at 37°C, fungi were cultured on PDA plates at 28°C, and colonies were observed and counted after overnight culture.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010537963A CN101967470B (en) | 2010-11-10 | 2010-11-10 | Cellulase stabilizing auxiliary agent composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010537963A CN101967470B (en) | 2010-11-10 | 2010-11-10 | Cellulase stabilizing auxiliary agent composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101967470A CN101967470A (en) | 2011-02-09 |
| CN101967470B true CN101967470B (en) | 2011-12-28 |
Family
ID=43546694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010537963A Expired - Fee Related CN101967470B (en) | 2010-11-10 | 2010-11-10 | Cellulase stabilizing auxiliary agent composition |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101967470B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881828A (en) * | 2017-10-17 | 2018-04-06 | 福建希源纸业有限公司 | A kind of methods of beating using biology enzyme |
| CN112746068B (en) * | 2019-10-31 | 2022-09-09 | 中国石油化工股份有限公司 | Method for improving stability of cellulase |
| CN114686466B (en) * | 2020-12-31 | 2023-07-28 | 中国石油化工股份有限公司 | Cellulase stabilizing synergist and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4272995B2 (en) * | 2001-12-18 | 2009-06-03 | 明治製菓株式会社 | Cellulase preparation containing reducing agent and fiber processing method |
| CN101381716B (en) * | 2008-10-23 | 2012-05-23 | 肇东市日成酶制剂有限公司 | Composite stabilizer for preserving liquid cellulase |
| CN101381717B (en) * | 2008-10-23 | 2010-08-25 | 肇东市日成酶制剂有限公司 | Composite stabilizer for preservation of liquid acid protease |
-
2010
- 2010-11-10 CN CN201010537963A patent/CN101967470B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101967470A (en) | 2011-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0494207B1 (en) | Thermostable purified cellulase from thermophilic bacterium acidothermus cellulolyticus | |
| Anand et al. | Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues | |
| Zhang et al. | Characterization of a thermophilic lignocellulose-degrading microbial consortium with high extracellular xylanase activity | |
| US10457925B2 (en) | Process for the production of cellulolytic and/or hemicellulolytic enzymes | |
| CN101967470B (en) | Cellulase stabilizing auxiliary agent composition | |
| Dhillon et al. | Bioproduction of hydrolytic enzymes using apple pomace waste by A. niger: applications in biocontrol formulations and hydrolysis of chitin/chitosan | |
| Sheng et al. | Bioaugmentation with Ruminiclostridium thermocellum M3 to enhance thermophilic hydrogen production from agricultural solid waste | |
| CN103789349A (en) | Method for increasing microbe conversion rate of bagasse by utilizing non-ionic surface active agent | |
| CN102159707A (en) | Family 6 cellulase with decreased inactivation by lignin | |
| CN101717765B (en) | A kind of compound enzyme preparation of cyclodextrin glucosyltransferase | |
| Weimer et al. | Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum | |
| CN103509730B (en) | Paenibacillus, breeding method and method for producing beta-glucanase by using same | |
| CN104428422A (en) | Process for the production of enzyme mixtures using liquid residues from biochemical conversion processes of lignocellulosic materials | |
| BRPI0705744B1 (en) | COMPOSITION OF ENZYMES, USE OF COMPOSITION IN ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE MATERIAL, PROCESS OF PRODUCTION OF ENZYMES THAT DEGRADES OF THE FRACTION OF POLYMACARIDES OF PROCESS | |
| CN107058448A (en) | Fermentation method of high-yield bacterial cellulose | |
| Marichamy et al. | Rapid production of cellulase-free xylanases by solventogenic Clostridia from rumen | |
| CN104560833A (en) | Basophilic micrococcus and alkaline xylanase produced from basophilic micrococcus and application | |
| Li et al. | Heterologous expression in Pichia pastoris and characterization of a β-glucosidase from the xylophagous cockroach Panesthia angustipennis spadica displaying high specific activity for cellobiose | |
| Florencio | Microrganismos produtores de celulases: seleção de isolados de Trichoderma spp | |
| Smith et al. | Isolation of mutants of Aspergillus awamori with enhanced production of extracellular xylanase and β-xylosidase | |
| CN104403980A (en) | Complex bacterial preparation and method for preparing cellulose by using complex bacterial preparation | |
| Xu et al. | Purification and characterization of an endo-1, 4-β-D glucanase from the cellulolytic system of the wood-boring marine mollusk Lyrodus pedicellatus (Bivalvia: Teredinidae) | |
| Ximenes et al. | Evaluation of a Hypocrea jecorina enzyme preparation for hydrolysis of Tifton 85 bermudagrass | |
| CN106011114A (en) | Fermentation liquor treatment method for increasing stability of pullulanase | |
| CN117363542A (en) | Semisynthetic culture medium for high-density culture of wine coccus and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20111228 Termination date: 20151110 |
|
| EXPY | Termination of patent right or utility model |