CN103805579B - 一种热稳定的淀粉酶突变体及其制备方法和应用 - Google Patents

一种热稳定的淀粉酶突变体及其制备方法和应用 Download PDF

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CN103805579B
CN103805579B CN201410056609.0A CN201410056609A CN103805579B CN 103805579 B CN103805579 B CN 103805579B CN 201410056609 A CN201410056609 A CN 201410056609A CN 103805579 B CN103805579 B CN 103805579B
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CN103805579A (zh
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吴敬
李祝
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Shandong Yellow Triangle Biotechnology Industry Research Institute Co ltd
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Jiangnan University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract

本发明公开了一种热稳定的淀粉酶突变体及其制备方法和应用，属于遗传工程领域。本发明以嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)淀粉酶为母本，在其N端区域与短肽通过PT-linker连接得到的突变体；短肽氨基酸序列如SEQIDNO.1所示。在此改造条件下，嗜热脂肪芽孢杆菌淀粉酶在95℃的半衰期由对照(突变前)例的4h提高到7h。利用此策略可以显著提高淀粉酶的热稳定性，为其工业化生产提供了基础。

Description

一种热稳定的淀粉酶突变体及其制备方法和应用
技术领域
本发明涉及一种淀粉酶突变体及其制备方法，特别是一种热稳定的淀粉酶突变体及其制备方法。
背景技术
α-淀粉酶，系统名称为α-1,4-葡萄糖-4-葡萄糖水解酶。α-淀粉酶的特点是在淀粉分子链中间将α-1,4糖苷键切断，不能水解α-1,6糖苷键，也不能水解紧靠1,6分支的α-1,4糖苷键，水解产物为可溶性糊精、低聚糖及少量麦芽糖、葡萄糖。α-淀粉酶具有相当大的工业应用价值，广泛用于酒精、有机酸、氨基酸及纺织等行业。热稳定淀粉酶生产菌株的获得主要通过筛选、诱变和酶分子改造获得。筛选的盲目性较大，不容易获得目的菌株。诱变包括：自然突变和诱变，自然突变的几率相当小，诱变的工作量较大且不可控出现负突变的几率较大。酶分子改造目的性强，针对酶分子具体结构分析进行改造，达到热稳定性提高的目的。
发明内容
本发明提供了一种热稳定性淀粉酶突变体，是淀粉酶N端区域与短肽通过PT-linker连接得到的突变体；短肽氨基酸序列如SEQIDNO.1所示。所述淀粉酶氨基酸序列如SEQIDNO.2所示。
所述PT-linker序列如SEQIDNO.3所示。
本发明还提供了一种制备所述淀粉酶突变体的方法，其技术方案如下：
1)据短肽和PT-linker序列，采用化学全合成的方法全合成后克隆到质粒pUC57中，构建重组质粒pUC57-P；
2)根据淀粉酶基因序列，采用化学全合成的方法全合成后克隆到质粒pET-20b(+)中，构建重组质粒pET-20b(+)-Amy；
3)针对已分析序列设计引物，将重组质粒pUC57-P中的短肽和PT-linker序列克隆到重组质粒pET-20b(+)-Amy中，构建含有短肽、PT-linker和淀粉酶片段的重组质粒pET-20b(+)-AmyP，将短肽融合到淀粉酶的N端，获得含有突变淀粉酶序列与短肽序列融合状态的重组载体；
4)将突变后重组载体转化大肠杆菌BL21(DE3)，诱导表达，获得突变体。
所述短肽氨基酸序列如SEQIDNO.1所示。
所述淀粉酶氨基酸序列如SEQIDNO.2所示。
所述PT-linker氨基酸序列如SEQIDNO.3所示。
所述淀粉酶突变体在降解、催化淀粉性基质中的应用也属于本发明要求保护的范围。
本发明提供的淀粉酶突变体的热稳定性好，在此改造条件下，嗜热脂肪芽孢杆菌淀粉酶在95℃的半衰期由对照(突变前)例的4h提高到7h。相对于采用筛菌或诱变等手段，缩短了酶学性质改造时间。将该热稳定性提高的淀粉酶突变体应用于淀粉液化等领域，可以在高温下高效降解淀粉，具有广阔的应用前景。
附图说明
图1：pET-20b(+)-AmyP质粒图谱
具体实施方式
实施例1：淀粉酶热稳定性提高突变分析和方法
通过对淀粉酶3D空间结构进行分析，确定区域A(催化区域)、区域B、区域C。根据淀粉酶的氨基酸序列及结构相关性，选用短肽与淀粉酶序列进行融合表达，提高其热稳定性。
据短肽和PT-linker序列，采用化学全合成的方法全合成后克隆到质粒pUC57中，构建重组质粒pUC57-P。同时根据嗜热脂肪芽孢杆菌的淀粉酶序列，采用化学全合成的方法全合成后克隆到质粒pET-20b(+)中，构建重组质粒pET-20b(+)-Amy。针对已分析序列设计引物，将重组质粒pUC57-P中的短肽和PT-linker序列克隆到重组质粒pET-20b(+)-Amy中，获得含有突变淀粉酶序列与短肽序列融合状态的重组载体pET-20b(+)-AmyP（图1）。突变后重组载体转化大肠杆菌BL21(DE3)，诱导表达，获得突变体。短肽氨基酸序列如SEQIDNO.1所示；淀粉酶氨基酸序列如SEQIDNO.2所示；PT-linker氨基酸序列如SEQIDNO.3所示。
实施例2：淀粉酶活力测定方法
DNS法测定淀粉酶酶活：
1)DNS试剂的配制：称取6.5g3,5-二硝基水杨酸溶于少量水中，移入1L容量瓶中，加入2mol/L氢氧化钠溶液262mL，再加入185g酒石酸钾钠及5g苯酚和5g无水亚硫酸钠，定容至1L，储存至棕色瓶中，放置于4℃冰箱待用。
2)葡萄糖标准曲线的制作：配制0.1g/L-1.0g/L不同浓度的葡萄糖溶液。取2mL不同浓度的葡萄糖与3mL的DNS溶液混合，放入沸水浴中，水浴7min。用冷水冷却，定容至15mL，A540测定吸光值。以葡萄糖浓度为横坐标，以吸光值为纵坐标，制作标准曲线。
3)酶活力单位定义：在pH6.0，温度70℃条件下，在1min降解可溶性淀粉产生1μmol还原糖物质(以葡萄糖计)所需要的酶量为1个酶活力单位(U)。
实施例3：淀粉酶热稳定性测定与分析
分别将融合前后的淀粉酶在含有20%甘油、pH6.0、95℃条件下处理，定期取样，采用实施例2的方法测定其残留酶活，通过对比发现，融合短肽后的淀粉酶在该测定条件下的半衰期从4h提高到7h，半衰期提高了1.7倍。该融合淀粉酶具有很好的热稳定性。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (3)

1.一种热稳定的淀粉酶突变体，其特征在于，短肽通过PT-linker与淀粉酶的N端连接得到的突变体；短肽氨基酸序列如SEQIDNO.1所示；所述淀粉酶氨基酸序列如SEQIDNO.2所示；所述PT-linker序列如SEQIDNO.3所示。
2.一种制备权利要求1所述淀粉酶突变体的方法，其特征在于，步骤如下：
1)根据短肽和PT-linker序列，采用化学全合成的方法全合成后克隆到质粒pUC57中，构建重组质粒pUC57-P；
2)根据淀粉酶基因序列，采用化学全合成的方法全合成后克隆到质粒pET-20b(+)中，构建重组质粒pET-20b(+)-Amy；
3)将重组质粒pUC57-P中的短肽和PT-linker序列克隆到重组质粒pET-20b(+)-Amy中，构建含有短肽、PT-linker和淀粉酶的重组质粒pET-20b(+)-AmyP，将短肽融合到淀粉酶的N端，获得含有淀粉酶序列与短肽序列融合状态的重组载体；
4)将步骤3)获得的重组载体pET-20b(+)-AmyP转化大肠杆菌BL21(DE3)，诱导表达，获得突变体。
3.权利要求1所述淀粉酶突变体在降解、催化淀粉基质中的应用。
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CN109234339B (zh) * 2018-11-22 2020-12-29 湖南汇升生物科技有限公司 一种高纯度结晶麦芽糖的生产方法
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WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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