CN113832134B - 一种糖用固定化酶的活力恢复的方法 - Google Patents
一种糖用固定化酶的活力恢复的方法 Download PDFInfo
- Publication number
- CN113832134B CN113832134B CN202111169687.8A CN202111169687A CN113832134B CN 113832134 B CN113832134 B CN 113832134B CN 202111169687 A CN202111169687 A CN 202111169687A CN 113832134 B CN113832134 B CN 113832134B
- Authority
- CN
- China
- Prior art keywords
- purified water
- activity
- sugar
- immobilized
- immobilized enzyme
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2431—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01009—Inulosucrase (2.4.1.9)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
一种糖用固定化酶活力恢复方法,通过纯净水清洗活力下降50%‑60%的糖用固定化酶,真空度负0.04‑负0.06Mpa下搅拌处理20‑30min,再壳聚糖吸附3‑5小时后用浓度为0.2‑0.5mol/L的乙酸调节使壳聚糖溶解、过滤分离、再次用纯净水清洗2‑3次,沥干水后,用铝复合膜包装封口,置于钴60辐照,剂量为2‑5KGy,然后置于温度2‑5℃放置24小时,即检测得到糖用固定化酶活力恢复到初始活力的77‑83%。
Description
技术领域
本发明涉及一种糖用固定化酶活力恢复的方法,具体为利用纯净水洗涤、负压处理、壳聚糖吸附、钴60辐照步骤获得活力恢复的方法。
背景技术
酶是一种活性蛋白,高效的生物催化剂,具有催化效率高。酶催化反应具有底物专一性、催化高效性、反应条件温和等优点,生物制品、食品工业 、环境保护、生物质能源、医药等广泛应用。由于液体游离酶在使用过程中存在稳定性差,适应温度范围窄、容易失活;酶反应与产品混在一起,难于分离,不利于重复利用,生产成本高。于是有了固定化酶(immobilized enzyme),是指酶被载体束缚在一定的空间范围内起催化作用,并能反复和连续使用的酶。固定化酶具有能重复多次地使用,产品容易分离、稳定性显著提高、反复可长期使用等。而固定化酶的载体为多孔、具有吸附性能如活性炭、高岭土、氧化铝、硅胶、微孔玻璃、多孔陶瓷、纤维素、火棉胶、CMC、DEAE-纤维素、DEAE-葡聚糖凝胶、壳聚糖、离子交换树脂等。酶固定后在使过程这些具有吸附功能载体在反应体系中吸附其它杂质、使固定化酶减少与底物接触降低其活力。特别糖用固定化酶在糖液中杂质中有各类蛋白质、胶质、色素、再加上糖本身容易引发微生物生长等导致糖用固定化酶活力更容易损失。固定化酶除固定化不牢固酶容易从载体脱落、反应中受到离子影响因素使固定化酶活力损失之外,就是载体吸附杂质、固定化酶染菌引起固定化酶活力降低。本发明是针对糖用固定化酶在使过程中杂质影响、染菌、离子影响导致糖用固定化酶活力降低后的活力恢复方法。
发明内容
解决糖用固定化酶生产过程中活力下降后,重新恢复活力,提高了生产效率、降低生产成本。本发明的目的在于提供一种糖用固定化酶活力恢复的方法,具有工艺合理、节能环保、恢复效果好、延长固定化酶使用寿命的优点。
本发明通过以下技术方案实现上述目的:一种糖用固定化酶活力恢复的方法,包括如下步骤:
(1)取活力下降50-60%的糖用固定化酶,用纯净水温度为37-42℃,电导率≤12us/cm的2-3倍重量的纯净水清洗2-3次;
(2)加入1.2-1.5倍重量的电导率≤12us/cm纯净水,在真空度负0.04-负0.06Mpa,温度37-42℃,转速=35-40r/min下搅拌20-30min,过滤得到滤物,用纯净水洗涤滤物2-3次;
(3)滤物加到2倍体积纯净水,调节温度至26-28℃,添加1.3-1.7%壳聚糖(W/W),转速=35-45r/min搅拌3-5小时后用浓度为0.2-0.5mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.6-1.8倍重量的纯净水,用0.1-0.3mol/L氢氧化钠溶液调节pH值为7.2-7.3,静置45-50min,过滤,滤物用水温度为30-33℃,电导率≤12us/cm纯净水清洗2-3次,沥干水,用铝复合膜包装封口;
(5)用钴60辐照,辐照剂量为2-5KGy;
(6)在温度2-5℃放置24小时,得到糖用固定化酶活力恢复到初始活力的77-83%。
本发明突出优点在于:
1.利用负压把固定化载体吸附的杂质重新解吸,释放出来,重新恢复载体性能,增加底物与酶接触面积,提高糖用固定化酶活力;
2.使用壳聚糖吸附糖用固定化酶的吸附的离子、金属等,同时壳聚糖溶液杀菌功能,减少离子、金属、细菌等对糖用固定化酶影响,重新提高了糖用固定化酶活力;
3.利用钴60辐照,由于辐射作用,对糖用固定化酶有一定杀菌作用。同时原来受到离子影响到糖用固定化酶结构变形的部酶发生移位、扭曲等情况,通过钴60低量辐射后变回原形,重新恢复活力。
具体实施方式
以下通过实施例对本发明的技术方案作进一步说明。
糖用固定化酶的活力检测均按本公司企业标准检测。
实施例1
所用纯净水电导率8us/cm,糖用固定化酶为固定化葡萄糖氧化酶。
(1)取活力下降60%的糖用固定化酶,用纯净水温度为37℃,用2倍重量的纯净水清洗2次;
(2)加入1.2倍重量的纯净水,在真空度负0.04Mpa,温度37℃,转速=35r/min下搅拌25min,过滤得到滤物,用纯净水洗涤滤物2-3次;
(3)滤物加到2倍体积纯净水,调节温度至28℃,添加1.6%壳聚糖(W/W),转速=40r/min搅拌2小时后用浓度为0.3mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.6倍重量的纯净水,用0.12mol/L氢氧化钠溶液调节pH值为7.2,静置50min,过滤,滤物用水温度为30℃,纯净水清洗2次,沥干水,用铝复合膜包装封口;
(5)用钴60辐照,辐照剂量为2KGy;
(6)在温度2℃放置24小时,测得固定化葡萄糖氧化酶活力恢复至初始活力的77%。
实施例2
本发明所述的一种糖用固定化酶活力恢复的方法的另一个实例,包括如下步骤:
所用纯净水电导率5us/cm,糖用固定化酶为固定化蔗糖转化酶。
(1)取活力下降60%的糖用固定化酶,用纯净水温度为40℃,用3倍重量的纯净水清洗3次;
(2)加入1.5倍重量的纯净水,在真空度负0.06Mpa,温度42℃,转速=40r/min下搅拌30min,过滤得到滤物,用纯净水洗涤滤物3次;
(3)滤物加到2倍体积纯净水,调节温度至26℃,添加1.3%壳聚糖(W/W),转速=35r/min搅拌3小时后用浓度为0.25mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.8倍重量的纯净水,用0.15mol/L氢氧化钠溶液调节pH值为7.3,静置45min,过滤,滤物用水温度为32℃,纯净水清洗2次,沥干水,用铝复合膜包装封口;
(5)用钴60辐照,辐照剂量为4KGy;
(6)在温度5℃放置24小时,测得到固定化蔗糖转化酶的活力恢复到初始活力的78%。
实施例3
本发明所述的一种糖用固定化酶活力恢复的方法的又另一个实例,包括如下步骤:
所用纯净水电导率10us/cm,糖用固定化酶为固定化果糖基转移酶。
(1)取活力下降53%的糖用固定化酶,用纯净水温度为38℃,用3倍重量的纯净水清洗2次;
(2)加入1.4倍重量的纯净水,在真空度负0.05Mpa,温度42℃,转速=35r/min下搅拌25min,过滤得到滤物,用纯净水洗涤滤物2次;
(3)滤物加到2倍体积纯净水,调节温度至27℃,添加1.5%壳聚糖(W/W),转速=44r/min搅拌4小时后用浓度为0.3mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.7倍重量的纯净水,用0.3mol/L氢氧化钠溶液调节pH值为7.3,静置50min,过滤,滤物用水温度为33℃,纯净水清洗3次,沥干水,用铝复合膜包装封口;
(5)用钴60辐照,辐照剂量为3KGy;
(6)在温度4℃放置24小时,测得固定化果糖基转移酶的活力恢复到初始活力的83%。
实施例4
本发明所述的一种糖用固定化酶活力恢复的方法的又再另一个实例,包括如下步骤:
所用纯净水电导率3us/cm,糖用固定化酶为固定化阿洛酮糖C3异构酶。
(1)取活力下降55%的糖用固定化酶,用纯净水温度为40℃,用2倍重量的纯净水清洗3次;
(2)加入1.3倍重量的纯净水,在真空度负0.06Mpa,温度40℃,转速=40r/min下搅拌25min,过滤得到滤物,用纯净水洗涤滤物3次;
(3)滤物加到2倍体积纯净水,调节温度至28℃,添加1.7%壳聚糖(W/W),转速=40r/min搅拌3小时后用浓度为0.2mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.5倍重量的纯净水,用0.2mol/L氢氧化钠溶液调节pH值为7.2,静置50min,过滤,滤物用水温度为32℃,纯净水清洗3次,沥干水,用铝复合膜包装封口;
(5)用钴60辐照,辐照剂量为5KGy;
(6)在温度5℃放置24小时,测得固定化阿洛酮糖C3异构酶的活力恢复到初始活力的79%。
Claims (1)
1.一种糖用固定化酶活力恢复的方法,其特征在于所述的糖用固定化酶为固定化蔗糖转化酶、固定化果糖基转移酶、固定化葡萄糖氧化酶、固定化阿洛酮糖C3异构酶,包括如下步骤:
(1)活力下降50-60%的糖用固定化酶,用2-3倍重量的纯净水,纯净水温度为37-42℃,电导率≤12us/cm清洗2-3次;
(2)加入1.2-1.5倍重量的纯净水,在真空度负0.04-负0.06Mpa,温度37-42℃,转速=35-40r/min下搅拌20-30min,过滤得到滤物,用纯净水洗涤滤物2-3次;
(3)滤物加到2倍体积纯净水,调节温度至26-28℃,添加1.3-1.7%壳聚糖(W/W),转速=35-45r/min搅拌3-5小时后用浓度为0.2-0.5mol/L的乙酸调节使壳聚糖溶解、过滤分离,滤物加入1.6-1.8倍重量的纯净水,用0.1-0.3mol/L氢氧化钠溶液调节pH值为7.2-7.3,静置45-50min,过滤,滤物用水温度为30-33℃,电导率≤12us/cm纯净水清洗2-3次,沥干水,用铝复合膜包装封口;
(4)置于钴60辐照,所用辐照剂量2-5KGy;
(5)在温度2-5℃放置24小时,得到糖用固定化酶活力恢复到初始活力的77-83%。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111169687.8A CN113832134B (zh) | 2021-10-08 | 2021-10-08 | 一种糖用固定化酶的活力恢复的方法 |
ZA2022/07827A ZA202207827B (en) | 2021-10-08 | 2022-07-14 | Method for recovering activity of immobilized enzyme for sugars |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111169687.8A CN113832134B (zh) | 2021-10-08 | 2021-10-08 | 一种糖用固定化酶的活力恢复的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113832134A CN113832134A (zh) | 2021-12-24 |
CN113832134B true CN113832134B (zh) | 2022-11-29 |
Family
ID=78968370
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111169687.8A Active CN113832134B (zh) | 2021-10-08 | 2021-10-08 | 一种糖用固定化酶的活力恢复的方法 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN113832134B (zh) |
ZA (1) | ZA202207827B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4250260A (en) * | 1979-08-22 | 1981-02-10 | Uop Inc. | Regeneration of an immobilized enzyme system |
JP2005013156A (ja) * | 2003-06-27 | 2005-01-20 | Fujimori Kogyo Co Ltd | 固定化物及びその再生方法 |
CN102286444A (zh) * | 2011-07-05 | 2011-12-21 | 上海交通大学 | 生物酶制剂的保藏方法 |
CN103224917A (zh) * | 2013-04-28 | 2013-07-31 | 北京凯因科技股份有限公司 | 一种恢复SAMe合成酶活力的方法 |
-
2021
- 2021-10-08 CN CN202111169687.8A patent/CN113832134B/zh active Active
-
2022
- 2022-07-14 ZA ZA2022/07827A patent/ZA202207827B/en unknown
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4250260A (en) * | 1979-08-22 | 1981-02-10 | Uop Inc. | Regeneration of an immobilized enzyme system |
JP2005013156A (ja) * | 2003-06-27 | 2005-01-20 | Fujimori Kogyo Co Ltd | 固定化物及びその再生方法 |
CN102286444A (zh) * | 2011-07-05 | 2011-12-21 | 上海交通大学 | 生物酶制剂的保藏方法 |
CN103224917A (zh) * | 2013-04-28 | 2013-07-31 | 北京凯因科技股份有限公司 | 一种恢复SAMe合成酶活力的方法 |
Non-Patent Citations (2)
Title |
---|
Extraction and reimmobilization of used commercial lipase from industrial waste;Nur Fathiah Mokhtar et al.;《International Journal of Biological Macromolecules》;20210205;第413-423页 * |
酶法合成头孢氨苄过程中固定化青霉素酰化酶酶活稳定性及其再生研究;安蔚;《中国优秀硕士学位论文全文数据库 基础科学辑》;20170615;摘要,第45-57页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113832134A (zh) | 2021-12-24 |
ZA202207827B (en) | 2022-10-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
FI85384B (fi) | Foerfarande foer hydrolysering av hemicellulosa med immobiliserade enzymer och produkt som omfattar ett immobiliserat hemicellulolytiskt enzym. | |
US9139856B2 (en) | Process for production of galactooligosaccharides (GOS) | |
WO2018228246A1 (zh) | 一种酶法制备谷胱甘肽的方法 | |
EP3255147B1 (en) | Immobilized cell and preparation method thereof | |
CN113832134B (zh) | 一种糖用固定化酶的活力恢复的方法 | |
FI79557C (fi) | Foerfarande foer isomerisering av glukos till fruktos. | |
US3201325A (en) | Process for the recovery of collagenase | |
US3386888A (en) | Resolution of racemic amino acids | |
CA1179283A (en) | SUCROSE MUTASE, IMMOBILISED SUCROSE MUTASE AND THE USE OF THIS IMMOBILISED SUCROSE MUTASE FOR THE PREPARATION OF ISOMALTULOSE (6-0-.alpha.-D- GLUCOPYRANOSIDO-D-FRUCTOSE) | |
CN111065644B (zh) | 一种制备高纯度nad的方法 | |
CN101270378B (zh) | 米格列醇中间体n-取代-1-脱氧野尻霉素衍生物的制备方法 | |
CA1127573A (en) | Method using glucoamylase immobilized on porous alumina | |
CN111410670B (zh) | 一种利用木质纤维素制备低聚糖的方法 | |
JP3632242B2 (ja) | パラチノースおよびトレハルロースの製造方法 | |
Su et al. | A novel method for continuous production of cyclodextrins using an immobilized enzyme system | |
EP0185379A2 (en) | A process for the production of purified glucose isomerase | |
CA2003767A1 (en) | Biocatalysts and processes for the manufacture thereof | |
CN104805159A (zh) | 一种提高α-葡萄糖基化丁香酚产量的方法 | |
CN108148826B (zh) | 一种β-葡萄糖苷酶的固定化方法 | |
KR800000241B1 (ko) | 고정화 포도당 이성화 효소의 제조방법 | |
CN107267564B (zh) | 一种ε-聚赖氨酸的生产方法 | |
CN117363674A (zh) | 一种小分子硫酸软骨素铁的制备方法 | |
CN100334221C (zh) | 一种直接法细胞酶反应生产丙烯酰胺水溶液的方法 | |
JPS5937959B2 (ja) | ブドウ糖濃度向上法 | |
CN110818753A (zh) | 一种结晶海藻糖母液循环利用方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |