CN104762334A - 酶催化反应生产过氧乙酸的方法 - Google Patents
酶催化反应生产过氧乙酸的方法 Download PDFInfo
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- CN104762334A CN104762334A CN201510176728.4A CN201510176728A CN104762334A CN 104762334 A CN104762334 A CN 104762334A CN 201510176728 A CN201510176728 A CN 201510176728A CN 104762334 A CN104762334 A CN 104762334A
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- Prior art keywords
- peracetic acid
- enzyme
- substrate
- ala
- hydrolysis reaction
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- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 title claims abstract description 145
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 20
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 13
- 230000002255 enzymatic effect Effects 0.000 title abstract 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 58
- 239000000758 substrate Substances 0.000 claims abstract description 31
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 18
- 108010093941 acetylxylan esterase Proteins 0.000 claims abstract description 8
- 108010080434 cephalosporin-C deacetylase Proteins 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims description 52
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- 238000000034 method Methods 0.000 claims description 52
- 238000006243 chemical reaction Methods 0.000 claims description 39
- 239000007788 liquid Substances 0.000 claims description 36
- URAYPUMNDPQOKB-UHFFFAOYSA-N triacetin Chemical compound CC(=O)OCC(OC(C)=O)COC(C)=O URAYPUMNDPQOKB-UHFFFAOYSA-N 0.000 claims description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 30
- 239000001087 glyceryl triacetate Substances 0.000 claims description 17
- 235000013773 glyceryl triacetate Nutrition 0.000 claims description 17
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- 241000894006 Bacteria Species 0.000 claims description 15
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- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
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- KMGOBAQSCKTBGD-DLOVCJGASA-N Ala-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CN=CN1 KMGOBAQSCKTBGD-DLOVCJGASA-N 0.000 description 2
- LNNSWWRRYJLGNI-NAKRPEOUSA-N Ala-Ile-Val Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O LNNSWWRRYJLGNI-NAKRPEOUSA-N 0.000 description 2
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Abstract
本发明公开了一种酶法生产过氧乙酸的方法,本方法以短链乙酸酯和过氧化氢为底物,由乙酰木聚糖酯酶过水解催化反应生成过氧乙酸。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种从具有过水解活性的乙酰木聚糖酯酶AXE(Genbank:KC292495)由乙酸酯底物生产过氧乙酸。
背景技术
过氧乙酸(PAA),别名过醋酸、过氧化乙酸、乙酰过氧化氢,是过氧有机酸中结构简单,合成容易,相对稳定性较好的高活性、强氧化性有机酸。过氧乙酸的氧化能力高于过氧化氢,可作为杀菌消毒剂、氧化剂、环氧化剂,其具有独特的作用,是一种广谱、高效、速效、廉价的灭菌剂,广泛应用于医疗器械消毒、灭菌以及环境、物表、空气等疫原地消毒和预防性消毒。(韩双战,过氧乙酸的合成和应用.河北化工2008(6):41-43.)由于其结构中活性氧的存在,除了用作杀菌消毒剂外,过氧乙酸在造纸纺织、化学合成、环境工程、能源工程、聚合物工业及仿生化学领域都有广泛的应用研究,尤其是作为有机合成中的环氧化剂以及高效杀菌剂,有其突出的优良性能,开发利用前景广阔。(张腾云,等;过氧乙酸的合成及工业应用研究进展.化工进展2007(26):194-197)
过氧乙酸的合成方法有很多种, 但目前工业上采用的方法主要是过氧化氢法和乙醛氧化法两种。过氧化氢法是采用醋酸与过氧化氢反应,在强酸的催化作用下,制得过氧乙酸。常使用硫酸作为催化剂,反应过程剧烈,且往往需要高浓度醋酸,得到的过氧乙酸为强酸性。乙醛氧化法是在乙醛氧化生成醋酸的基础上,通过改变乙醛氧化的条件,降低反应温度,获得过氧乙酸。但此反应较复杂,要得到较高的转化率和过氧乙酸得率, 需加入适当的催化剂。且由于过氧乙酸具有强氧化性和腐蚀性,遇明火、高热会引起燃烧***;与还原剂接触、遇金属粉木有燃烧***危险,过氧乙酸的包装和运输及储存都有严格要求。(刘吉起,过氧乙酸的性质、制备和应用.河南预防医学杂志2004(03):171-173)
因此,人们开始积极探索生物法生产过氧乙酸。酶法或者微生物法生产过氧乙酸的优点在于反应迅速、温和,不需要高浓度过氧化氢或底物,可以解决运输和储存等问题。酯酶和脂肪酶,其能催化酯水解,据报道常有与非血红素卤代过氧化物重叠的催化活性,能在过氧化氢存在下催化过氧化反应,产生过氧羧酸。反应如下:
例如, O.Kirk等(Biocatalysis,11:65-77(1994)) 研究了水解酶(脂肪酶、酯酶和蛋白酶)催化酰基底物与过氧化氢的过水解以形成过氧羧酸;近期已报道几种蛋白酶和脂肪酶组合原位产生浓度适于用作消毒剂和/或商业漂白剂的过酸(例如过乙酸)(美国专利申请11/413,246和11/588,523)。然而,大多数已知的利用酶生物催化生产的过氧乙酸浓度较低,不具备有效的应用价值。
实现生物法制备过氧乙酸,最重要的是获得一种易于获取、高活性、高表达的酶,才能达到生产效率高、反应迅速灵敏的要求,进而实现大规模生产。然而,此问题仍然是制约生物法制备过氧乙酸的关键问题。
发明内容
发明人发现,具有自主知识产权(专利申请号CN201210403666.2)的乙酰木聚糖酯酶AXE具有过水解活性,基于此,提出了本发明方案。因此,发明人要求将专利申请CN201210403666.2的内容一并并入本申请,包括申请中乙酰木聚糖酯酶的氨基酸序列SEQ ID NO:2;具有活性的同源三聚体、六聚体蛋白结构;以及基因工程菌大肠杆菌(Escherichia coli)BL21-pET-Cah的构建过程,以及构建获得的菌株。
本发明所要解决的技术问题在于,获得一种易于获取、高活性、高表达的酶,来进行过氧乙酸的酶法生产。
本发明的技术目的在于提供该酯酶在过水解反应中的应用。具体的,可用该酶催化过水解反应,更具体的,在过氧化氢存在情况下,可用该酶催化短链乙酸酯生成过氧乙酸。
具体的,本发明的技术方案包括:
具有SEQ ID NO:1 代表的氨基酸序列的乙酰木聚糖酯酶在过水解反应中的应用。
其中,本申请中SEQ ID NO:1 代表的氨基酸序列与中国专利申请CN201210403666.2中所述SEQ ID NO:2中代表的氨基酸序列一致。首先,发明人发现了该酶具有过水解活性,能够在过氧源存在时催化酰基底物生成过氧酸;其次,对于过水解反应应该理解为,在具有过氧源,如过氧化氢时,酶将酰基底物催化生成过氧酸。
在优选的反应中,该酶可以在过氧化氢存在条件下催化乙酰基底物生成过氧乙酸。
一种酶催化生产过氧乙酸的方法,以乙酰基底物和过氧化氢为底物,将具有SEQ ID NO:1 代表的氨基酸序列的乙酰木聚糖酯酶与底物接触进行过水解反应生成过氧乙酸。
本发明所述的方法,其中,可以以含该酯酶蛋白质的载体为酶催化剂,与底物接触进行过水解反应生成过氧乙酸。
对于以含该蛋白质的载体为酶催化剂,可以理解为,可以将上述蛋白质在表达体系中表达后纯化、固定化;也可以将上述蛋白质表达后并将表达的菌株破碎,从而获得粗酶液,将粗酶液与底物反应以获取目标产物,或者将粗酶液或者全细胞固定化后将底物与之接触并反应获取目标产物。
本发明所述的方法,所述乙酰基底物选自乙酸乙酯、甘油一乙酸酯、甘油二乙酸酯、甘油三乙酸酯中的一种。
本发明所述的方法,其中,在所述过水解反应体系中,以毫克每毫升计,所述乙酰基底物:过氧化氢:酶的数量比例关系为1:0.5-8.5:0.015-0.075。
对于该比例关系应理解为,有底物种类存在满足的情况下即可实现本发明,而本发明提供的比例为优选的比例。
本发明所述的方法,其中,所述过水解反应的反应条件为:pH值6.5-9.5,优选为pH 8.0。
本发明所述的方法,其中,所述过水解反应的反应时间为5分钟到约2小时之内,优选为5分钟之内。
本发明所述的方法,其中,所述过水解反应的温度为20-37℃,优选20℃。
对于本发明中过水解的时间、温度、pH值的理解应理解为,有底物种类存在满足的情况下即可实现本发明,而本发明提供的条件为优选条件。
本发明所述的方法,其中,将还包括依据所述蛋白质的编码或氨基酸序列,将蛋白质表达获取粗酶液的过程。
本发明所述的方法,其中,依次包括如下步骤:
(1)以大肠杆菌(Escherichia coli)BL21/pET-CAH,为出发菌株进行种子培养;其中该大肠杆菌保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2012408;其中,种子培养培养基为LB培养基;培养条件为:250 mL三角瓶,装液量50 mL,培养温度37℃,摇床转速200 r/min,培养12 h;另外,经破壁获取的粗酶液,还可以通过固定化来进一步进行催化反应。
本发明的有益效果在于,为生物法催化合成过氧乙酸提供了一种全新的酶制剂。
另外,采用本发明的方案可以获取比现有生物法制得的更高的过氧乙酸终浓度。
附图说明
图1为对硝基苯酚(p-NP)浓度-OD405吸光值标准曲线。
图2 为牛血清蛋白(BSA)浓度-OD595吸光值标准曲线。
图3 为过氧乙酸(PAA)浓度-OD405吸光值标准曲线。
图4 为最适重组酯酶AXE用量优化实验结果。
图5 为最适甘油三乙酸酯浓度优化实验结果。
图6 为最适过氧化氢浓度优化实验结果。
图7 为最适pH优化实验结果。
具体实施方式
下面结合实施例对本发明做进一步说明。所列的实施例仅作阐示之用,并表明本发明的精神和范围并非限于此中的细节及其修改案。
本发明所使用的菌种来源:
基因工程菌(Escherichia coli)BL21/pET-CAH为本实验室所有,并保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2012408。该菌种的具体构建过程,以及酶的鉴定过程参见专利CN 102952787 A。
实施例1 本实施例说明酯酶的诱导表达方法。
利用基因工程菌诱导表达酯酶的具体方案如下:
(1) 出发菌株:大肠杆菌(Escherichia coli)BL21/pET-CAH;
(2) 种子培养:
培养基:LB培养基:酵母粉 5g/L,蛋白胨10 g/L,氯化钠10 g/L;
培养条件:250 mL三角瓶,装液量50 mL,培养温度37℃,摇床转速200 r/min,培养12 h;
(3) 发酵培养:
培养基组成:LB培养基:酵母粉 5g/L,蛋白胨10 g/L,氯化钠10 g/L;
培养条件:接种量1.5% (v/v),发酵温度37 ℃,当OD达到0.6~0.8时,加异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导,IPTG的终浓度1.0 mmol/L,摇床转速180~250 r/min,发酵2-10 h。
(4) 粗酶液的获取:
取出发酵液,8000rpm离心3min,加入1/8 原发酵液体积的Tris-HCl(pH 7.0),浓缩8倍;超声破碎400W、3s 工作时间,7s 间隙时间,超声4min (24次);离心6500 rpm、10 min。上清即为粗酶液。
(5) 酶活力的测定:
使用对硝基苯酚乙酸酯为底物,1个酶活单位(U)定义为:在一定反应条件下,每分钟释放1μL p-NP所需的酶量。
标准曲线制作:A溶液的配制:50 mmol/L的磷酸缓冲(pH7.0),其中含有0.6% Triton X-100和0.1%的***树胶;B溶液的配制:准确称取0.0139 g pNP标准物质,用A溶液溶解,容量瓶定容至250 mL,配成标准物质浓度为1 μmol/mL的B溶液。采用A溶液对B溶液进行梯度稀释,配成不同浓度的pNP溶液。在酶标板中加入10 μL缓冲液,然后依次加入不同浓度的pNP标准溶液各240 μL,对照中加入A溶液。用酶标仪测定405 nm波长处吸光值。以pNP的浓度作为横坐标,以吸光值作为纵坐标,作标准曲线(图1)。
测定步骤如下:在反应体系中加入240 μL 底物溶液(A溶液与B溶液按9:1混合),10 μL 适当稀释的酶液,置于水浴锅中,温度为40 ℃,反应时间10 min;使用酶标仪在405 nm处测定吸光值。实验设三个平行,取平均值,并使用10 μL 蒸馏水最为对照组。p-NP标准曲线及方程如图1。
测定结果显示:100倍稀释的酶液,A405nm下吸光值分别为:1.449、1.482、1.509,通过标准曲线(图1)计算,对应活性为224.96U/mL。
(6) 蛋白量的测定
使用Brandford法测定蛋白量。
蛋白标曲的制作:配制考马斯亮蓝溶液:100 mg 考马斯亮蓝G-250溶于50 mL 95 % 乙醇中,再加入100 mL 85 %(v/v) H3PO4,最后用蒸馏水稀释至1 l,滤纸过滤后可使用;配制0.1 g/l BSA:称取0.01 g BSA,溶于10 mL 蒸馏水中,使用前用生理盐水配制成0.01、0.02 、0.03、0.04、0.06、0.08 mg/mL;取7个2 mL离心管,编好号(0,1,2,3,4,5,6),在1~6号离心管中分别加入上述稀释后各浓度BSA溶液0.3 mL,最后在每支离心管中加入1.2 mL 考马斯亮蓝溶液。在0号离心管中1.2 mL 考马斯亮蓝溶液和0.3 mL生理盐水。各管混合后后,以0号管为对照测定A595。测得的标准曲线如图2。
蛋白的检测具体步骤如下:首先以牛血清蛋白(BSA)标样的浓度对OD595吸光值作标准曲线方程,如图2。然后去适当稀释的酶液300μL ,加入1.2mL考马斯亮蓝工作液,混匀,室温放置15min,与595nm下测定吸光值。每组做两个平行样,以蒸馏水为空白。
测定结果显示稀释5倍的酶液反应后的OD595吸光值为,0.252、0.277。按照图2的标准曲线计算后,蛋白浓度为:0.16 mg/mL 。因此,工程菌发酵液的乙酰基木聚糖酯酶粗酶液的比酶活力为1406 U/mg。
实施例2 本实施例说明替换基因工程菌表达载体为pET-28a,构建新基因工程菌E. coli BL21pET28a-Cah并对该酯酶进行5L罐规模诱导表达的方法。
(1)基因Cah的克隆:
根据专利申请号CN201210403666.2中公布的Cah基因(Genbank:KC292495),序列设计合引物:
P1:5’-CATGCCATGGGCATGCAATTATACGACT-3’。
P2:5’-CCCTCGAGGCCTTTCAGATGCGCTT-3’。
引物两端分别引入限制性酶切位点NcoI和XhoI,依照质粒提取试剂盒(OMEGA公司)的说明提取基因工程菌(Escherichia coli)BL21/pET-CAH质粒,以基因工程菌(Escherichia coli)BL21/pET-CAH质粒DNA为模板完成PCR反应;
PCR的8管反应体系是:Buffer 22.5μL,ddH2O 141.3μL,MgCl2 13.5μL,dNTP-mix 18μLL,引物P1 4.5μL,引物P2 4.5μL,Taq酶 2.7μL;混合均匀后,分成8管,再向每管加入2μL模板DNA;PCR反应条件:94 ℃预变性 4 min;进行30个循环反应:94 ℃变性45 s,54.1 ℃退火30 s,72 ℃延伸60 s;最后72 ℃保温10 min,反应结束后4 ℃保存;经PCR纯化试剂盒(TaKaRa公司)纯化后,NcoI和XhoI酶切重组回收产物,回收957 bp片段,NcoI和XhoI酶切pET28a载体,回收大片段产物,将双酶切产物在T4DNA连接酶作用下连接获得重组质粒pET28a-Cah。
(2) 重组基因工程菌的获得:
将重组质粒pET28a-Cah转化E. coli BL21 (DE3),涂布含50 μg/mL的卡那霉素平板,挑取阳性转化子,并进行菌落PCR鉴定,得到重组E.coli,命名为E.coli BL21pET28a-Cah,提取E.coli BL21pET28a-Cah的重组质粒,进行测序验证。
(3) 重组基因工程菌E. coli BL21pET28a-Cah 5L罐规模诱导表达的方法
种子培养:
培养基:LB培养基:酵母粉 5g/L,蛋白胨10 g/L,氯化钠10 g/L;
培养条件:250 mL三角瓶,装液量50 mL,培养温度37 ℃,摇床转速200 r/min,培养12 h;
发酵培养:
培养基组成:自诱导培养基:酵母粉 25g/L,胰蛋白胨15 g/L,氯化钠10 g/L,甘油(v/v)0.1%,葡萄糖(w/v)0.2%,乳糖(w/v)0.2%;
培养条件:5L发酵罐,装液量为3L,接种量3.3% (v/v),发酵温度30 ℃,pH7.5,搅拌转速 200 r/min,通气量1.5 vvm发酵16 h。
(4) 粗酶液的获取:参照实例1中粗酶液的获取方法
(5) 酶活力的测定:参照实例1中检测方法进行鉴定。结果表明,经过16小时5L罐自诱导表达,可获得酯酶活力为2.28×104U/ml,通过替换表达载体为pET-28a,并进行5L罐自诱导表达该酯酶(替换昂贵的IPTG诱导剂),说明可以进一步降低生产酯酶培养基成本,从而降低酶法生产过氧乙酸的成本,更有利于实现工业化生产。
实施例3 本实施例说明以不同短链乙酸酯和过氧化氢为底物,重组酯酶催化生产过氧乙酸及过氧乙酸的检测。
操作步骤:
(1)按照实施例1中的方法获取酶液;
(2)利用酯酶催化制备过氧乙酸的反应体系和操作流程:短链乙酸酯底物(乙酸乙酯、甘油一乙酸酯、甘油二乙酸酯、甘油三乙酸酯)0.1g,过氧化氢100 μL(1mol/l),酯酶AXE 30μL (0.3g/ml),0.1mol/l pH7.4磷酸盐缓冲,共1 mL反应体系。其中空白对照中将酶液换成等量缓冲液;
(3)每个条件下的反应做3个平行,在反应5min、10min、15min时取样;
(4)产物的处理:反应结束后离心12000rpm,2min后取上层清液,即为反应产物;
(5)进行过氧乙酸检测。采用2 ,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)的氧化来检测过氧乙酸的浓度。将100μL 经稀释后的反应产物与100μL 0.5g/l ABTS溶液和50μL 1.5mol/l的含有0.03mg/ml 碘化钾的乙酸溶液混合于96孔板中,室温反应10min后,测量其在405nm处吸光度。
过氧乙酸标曲的制作:配制0.5g/l ABTS溶液(避光)以及1.5mol/l的含有0.03mg/ml 碘化钾的乙酸溶液:在96孔板编号1~10中依次加入0μL、5μL、10μL、15μL、20μL、25μL、30μL、35μL、40μL、50μL ABTS溶液,100μL、95μL、90μL、85μL、80μL、75μL、70μL 、65μL、60μL、50μL ddH2O,在编号1~10中加入50μL 1.5mol/l的含有0.03mg/ml 碘化钾的乙酸溶液,以及100μL标准过氧乙酸液,混合均匀后室温反应10min,以0号管为对照测定A405。测得的标准曲线如图3。
实验结果如下:25℃下反应5min后所获得的过氧乙酸浓度如表1所示,甘油三乙酸酯作为底物产生的过氧乙酸浓度最高,因此选择甘油三乙酸酯作为底物。
表1. 不同乙酸酯底物生产过氧乙酸浓度
实施例4本实施例说明以甘油三乙酸酯和过氧化氢为底物,重组酯酶催化生产过氧乙酸的条件优化
1.重组酯酶AXE用量的优化步骤:
(1) 按照实施例1中的方法获取酶液;
(2) 反应体系及条件:对重组酯酶AXE用量进行优化,1ml反应体系中含有250mmol/l甘油三乙酸酯,1mol/l过氧化氢在25℃、pH:7.4条件下反应5min;
(3) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明:如图4所示,随着酯酶的量的增加,催化生产的过氧乙酸的量也在增加,当酯酶增加到0.30mg/ml时,生成的过氧乙酸的量增加缓慢,因此从实际应用与成本考虑,该反应体系中重组酯酶的用量最优为0.30mg/ml,可产生8259ppm过氧乙酸。
2.甘油三乙酸酯浓度的优化。
操作步骤:
(1) 按照实施例1中的方法获取酶液;
(2) 反应体系及条件:对甘油三乙酸酯浓度进行优化,1ml反应体系中含有重组酯酶AXE 0.3mg/ml,1mol/l过氧化氢在25℃、pH:7.4条件下反应5min;
(3) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明:如图5所示,随着甘油三乙酸酯的浓度从100mmol/l增加到300mmol/l,生产的过氧乙酸的量随之增加,但当甘油三乙酸酯浓度高于300mmol/l时,过氧乙酸的量并没有明显增加,因此该反应体系中甘油三乙酸酯的浓度最优为300mmol/l,可产生9613ppm过氧乙酸。
3.过氧化氢浓度的优化
操作步骤:
(1) 按照实施例1中的方法获取酶液;
(2) 反应体系及条件:对过氧化氢浓度进行优化,1ml反应体系中含有300mmol/l甘油三乙酸酯,重组酯酶AXE 0.3mg/ml在25℃、pH:7.4条件下反应5min;
(3) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明:如图6所示,随着过氧化氢浓度的增加,催化生产的过氧乙酸的量先增加后减少,这可能是由于高浓度的过氧化氢导致酯酶的快速失活引起的。该反应体系中过氧化氢浓度最优为1mol/l,可产生9613ppm过氧乙酸。
4.温度的优化
操作步骤:
(1) 按照实施例1中的方法获取酶液;
(2) 反应体系及条件:不同温度下,1ml反应体系中含有300mmol/l甘油三乙酸酯,重组酯酶AXE 0.3mg/m,1mol/l过氧化氢在pH:7.4条件下反应5min;
(3) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明,如表2所示,20℃时,生产的过氧乙酸的量达到最高为10481ppm,具有显著的应用价值。
表2. 反应温度的优化
反应温度 | 过氧乙酸 ppm |
20℃ | 10481.63 |
25℃ | 9818.63 |
30℃ | 9730.00 |
37℃ | 8407.54 |
5.pH的优化操作步骤:
(1) 按照实施例1中的方法获取酶液;
(2) 反应体系及条件:不同pH条件下,1ml反应体系中含有300mmol/l甘油三乙酸酯,重组酯酶AXE 0.3mg/ml,1mol/l过氧化氢在20℃条件下反应5min;
(3) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明,如图7所示,pH8.0时,生产的过氧乙酸的量达到最高为11385ppm。
实施例5 本实施例说明固定化AXE酯酶生产过氧乙酸的步骤。
(1) 按照实施例1中的方法获取酶液;
(2) 重组酯酶AXE的固定化:选择聚丙烯酰胺树脂,利用戊二醛交联法对重组酯酶进行固定化,获得固定化AXE酯酶,对对硝基苯乙酸酯的活力为:17.5U/g
(3) 反应体系及条件:1ml反应体系中含有300mmol/l甘油三乙酸酯,1mol/l过氧化氢,0.5g固定化酶,在pH 8.0、20℃条件下反应5min;
(4) 按照实施例3中的方法获取过氧乙酸样品并检测。
结果表明:0.5g固定化后的重组酯酶AXE可产生2678ppm的过氧乙酸。相比于游离酯酶AXE,更便捷可回收使用。重复10次后产生的过氧乙酸为初次反应的72%左右。
本发明提供了以下方法,该方法用具有过水解活性的乙酰木聚糖酯酶AXE(游离状态或固定化状态),在过氧化氢(浓度至少200mmol/l)存在下,在酸性至中性反应条件下,从合适的短链乙酸酯(包括甘油酯)原位产生浓过氧乙酸水溶液。
序列表
<110> 南京工业大学
<120> 酶催化反应生产过氧乙酸的方法
<130> xb15041502
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<170> PatentIn version 3.3
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Claims (10)
1.具有SEQ ID NO:1 代表的氨基酸序列的乙酰木聚糖酯酶在过水解反应中的应用。
2.一种酶催化生产过氧乙酸的方法,以乙酰基底物和过氧化氢为底物,其特征在于,以权利要求1所述的乙酰木聚糖酯酶为酶催化剂,与底物接触进行过水解反应生成过氧乙酸。
3.根据权利要求2所述的方法,其特征在于,以含权利要求1所述的乙酰木聚糖酯酶的载体为酶催化剂,与底物接触进行过水解反应生成过氧乙酸。
4.根据权利要求2或3所述的方法,其特征在于,所述乙酰基底物选自乙酸乙酯、甘油一乙酸酯、甘油二乙酸酯、甘油三乙酸酯中的一种。
5.根据权利要求4所述的方法,其特征在于,在所述过水解反应体系中,以毫克每毫升计,所述乙酰基底物:过氧化氢:酶的数量比例关系为1:0.5-8.5:1.5-75。
6.根据权利要求2或3所述的方法,其特征在于,所述过水解反应的反应条件为:pH值6.5-9.5,优选为pH 8.0。
7.根据权利要求2或3所述的方法,其特征在于,所述过水解反应的温度为20-37℃,优选20℃。
8.根据权利要求2或3所述的方法,其特征在于,所述过水解反应的反应时间为5分钟到约1小时之内,优选为5分钟之内。
9.根据权利要求2所述的方法,其特征在于,将还包括依据所述蛋白质的编码或氨基酸序列,将所述乙酰木聚糖酯酶表达并获取粗酶液的过程。
10.根据权利要求9所述的方法,其特征在于,依次包括如下步骤:
(1)以大肠杆菌(Escherichia coli)BL21/pET-CAH,为出发菌株进行种子培养;其中该大肠杆菌保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2012408;其中,种子培养培养基为LB培养基;培养条件为:250 mL三角瓶,装液量50 mL,培养温度37℃,摇床转速200 r/min,培养12 h;
(2) 发酵培养,发酵培养基组成为液体LB培养基;培养条件为,按照体积比为1.5%接种量接种种子培养液,发酵温度37 ℃,当OD达到0.6~0.8时,加异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导,IPTG的终浓度1.0 mmol/L,摇床转速180~250 r/min,发酵2-10 h;
(3) 粗酶液的获取,取出发酵液,8000rpm离心3min,加入1/8 原发酵液体积的Tris-HCl(pH 7.0),浓缩8倍;超声破碎400W、3s 工作时间,7s 间隙时间,超声4min;离心6500 rpm、10 min,上清即为粗酶液。
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