CN100593568C - 一种高效壳聚糖降解菌芽孢杆菌及其产生的壳聚糖酶 - Google Patents

一种高效壳聚糖降解菌芽孢杆菌及其产生的壳聚糖酶 Download PDF

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CN100593568C
CN100593568C CN200510048372A CN200510048372A CN100593568C CN 100593568 C CN100593568 C CN 100593568C CN 200510048372 A CN200510048372 A CN 200510048372A CN 200510048372 A CN200510048372 A CN 200510048372A CN 100593568 C CN100593568 C CN 100593568C
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chitosan
bacillus
enzyme
highly effective
chitoanase
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CN1995325A (zh
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肖湘
王凤平
汤熙翔
游志勇
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CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION
Third Institute of Oceanography SOA
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CHINA OCEAN MINERAL RESOURCES R&D ASSOCIATION
Third Institute of Oceanography SOA
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Abstract

本发明公开得到了一株壳聚糖降解菌——芽孢杆菌(Bacillus sp.)，该菌株能够非诱导性分泌一种胞外壳聚糖酶。经过对该菌株的发酵上清进行酶活性测定，该酶的最适反应温度为65℃，最适pH为6.0，纸层析和质谱分析结果显示该酶的最终酶解产物为壳聚二糖和壳聚三糖。

Description

一种高效壳聚糖降解菌芽孢杆菌及其产生的壳聚糖酶
技术领域：
本发明涉及一种筛选得到的一株高效壳聚糖降解菌——芽孢杆菌，该菌分泌一种壳聚糖酶，降解壳聚糖得到壳聚二糖和壳聚三糖。
背景技术：
壳聚糖为葡萄糖胺以β-1，4糖苷键连接而成的直链聚合物。它可以看作是几丁质脱乙酰的衍生物。广泛存在于接合菌和植物病原菌的细胞壁中，在绿藻和真菌以及一些人体病原菌的细胞壁中也有少量存在。壳聚糖酶(EC3.2.1.99)能够降解壳聚糖的β-1，4糖苷键，可分为内切酶和外切酶。壳聚糖内切酶能够随机切割多糖链(n≥4)得到分子量不同的壳聚寡糖。许多微生物能够分泌壳聚糖酶，降解壳聚糖为生长提供所需的碳源和氮源。
壳聚糖主要是指脱乙酰度大于80％的几丁质衍生物或即乙酰化低于20％的多糖胺链。研究发现低聚壳聚多糖具有增强植物病原抵抗力和诱导上皮细胞生成等多种功效；而最近的研究表明：壳聚六糖具有很强的抗肿瘤作用。目前，低聚糖的生产主要采用水力切割和酸水解等物理化学方法。这些方法具有生产力低下，产物为多种混合物等不易克服的缺点。
发明内容：
本发明的目的在于筛选得到一株高效壳聚糖降解菌——芽孢杆菌，该菌株所分泌的壳聚糖酶能够降解壳聚糖得到壳聚二糖和壳聚三糖。
我们从东太平洋深海沉积物中，分离得到一株具有较高的壳聚糖酶活性的芽孢杆菌。经鉴定该菌株为芽孢杆菌(Bacillus sp.)。经研究，该菌株的盐度适应范围为0-8％，最适培养基为没有添加NaCl的2216E培养基，其最适的生长温度为30℃。该菌株已提供给中国典型培养物保藏中心保藏，编号为CCTCC NO：M205103。
该菌株具有非诱导产酶的特点，低浓度(0.005％)的壳聚糖并不影响其产酶效率，但当培养基中的壳聚糖浓度增加到0.1％时，能够使得该菌株的产酶时间延迟，以及其产酶量具有较为明显的下降。
附图说明：
图1：TLC分析。M为Marker。不同反应时间在图的上方标识
图2：MS图：a，5分钟；b，60分钟；c，2天
具体实施方式：
实施例1：
细菌的筛选：壳聚糖酶平板的制备：通过含有0.1％壳聚糖的基本培养基的平板上不同菌落周围的出圈情况筛选所需的菌株。并通过16S rDNA进行初步的鉴定。
细菌产酶情况的研究：将所筛得的细菌接入到含0.1％壳聚糖的基本培养液中，30℃摇培过夜。浓缩上清以及破碎细胞，并将浓缩后的上清液和细胞破碎液加入含0.01％壳聚糖的McIlvaine缓冲液中，37℃反应2小时并用DNS的方法测定反应体系中还原糖的含量。
实施例2：
产酶性质的研究：通过在添加有壳聚糖和不加壳聚糖的基本培养液两种不同条件下菌株的生长情况，以及将发酵上清浓缩后作为粗酶进行酶活性验证，其结果显示该菌株为非诱导产酶。
实施例3：
酶活力测定：将发酵上清浓缩后作为粗酶，通过不同的PH梯度酶解产物即还原糖产生量的测定得出该酶的最适反应PH为PH6.5；再通过不同的温度梯度还原糖的产生效率得出该酶最适合的反应温度为60℃。
实施例4：
酶解产物的分析：通过TLC纸层析的方法，对不同时间段酶解产物进行展层分析。结果显示反应时间为5分钟时，反应体系中以多糖为主，随着反应的进行，多糖的含量逐渐减少，直至反应时间为60分钟后，反应体系中只有二糖、三糖和四糖；在酶反应持续两天后，体系中只剩下二糖和三糖，并且随着酶反应时间的增加，反应产物并不发生改变。
另一方面，通过不同酶反应时间产物的质谱分析显示的结果与TLC的结果一致。因此，可以得出该酶最终的反应产物为壳聚二糖和壳聚三糖，最小的识别底物为壳聚四糖，但降解的效率较低。

Claims (4)

1、一种芽孢杆菌(Bacillus sp.)，保藏号为：CCTCC NO：M205103。
2、如权利要求1所述的芽孢杆菌，其特征在于能够分泌胞外壳聚糖酶，降解壳聚糖得到二糖和三糖。
3、如权利要求1所述的芽孢杆菌，其特征在于能够非诱导性分泌胞外壳聚糖酶。
4、一种降解壳聚糖生产二糖和三糖的方法，其特征在于利用权利要求2所述的芽孢杆菌分泌胞外壳聚糖酶，以内切的方式降解壳聚糖最终产生壳聚二糖和壳聚三糖。
CN200510048372A 2005-12-31 2005-12-31 一种高效壳聚糖降解菌芽孢杆菌及其产生的壳聚糖酶 Expired - Fee Related CN100593568C (zh)

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CN101382520B (zh) * 2008-09-28 2011-12-21 湖南师范大学 利用质谱技术对苏云金杆菌菌株进行分类鉴定的方法
CN107129939A (zh) * 2016-02-29 2017-09-05 株式会社可伶格林 生产壳聚糖分解酶的微生物和利用其的植物栽培用组合物及栽培方法
CN106754829B (zh) * 2016-12-06 2020-10-16 鲁东大学 一种利用芽孢杆菌hs17发酵生产壳聚糖酶的方法及其应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Properties of Chitosanase from Bacillus cereus S1. Masahiro Kurakake等.CURRENT MICROBIOLOGY,Vol.40 . 2000
Properties of Chitosanase from Bacillus cereus S1. Masahiro Kurakake等.CURRENT MICROBIOLOGY,Vol.40. 2000 *
Some Pathogenesis-Related Proteins Are ChitosanaseswithLytic Activity Against Fungal Spores. Jean Grenier, Alain Asselin.MOLECULAR PLANT-MICROBE INTERACTIONS,Vol.3 No.6. 1990
Some Pathogenesis-Related Proteins Are ChitosanaseswithLytic Activity Against Fungal Spores. Jean Grenier, Alain Asselin.MOLECULAR PLANT-MICROBE INTERACTIONS,Vol.3 No.6. 1990 *
壳聚糖酶解的研究. 黄永春等.化工进展，第21卷第6期. 2002
壳聚糖酶解的研究. 黄永春等.化工进展，第21卷第6期. 2002 *
曲霉CJ22-326 内切壳聚糖酶的分离纯化和性质. 陈小娥等.无锡轻工大学学报，第23卷第3期. 2004
曲霉CJ22-326内切壳聚糖酶的分离纯化和性质. 陈小娥等.无锡轻工大学学报，第23卷第3期. 2004 *

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