CN1560226A - 用Alteromonas sp.nov.SY37-12菌株生产琼胶酶 - Google Patents

用Alteromonas sp.nov.SY37-12菌株生产琼胶酶 Download PDF

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CN1560226A
CN1560226A CNA2004100236561A CN200410023656A CN1560226A CN 1560226 A CN1560226 A CN 1560226A CN A2004100236561 A CNA2004100236561 A CN A2004100236561A CN 200410023656 A CN200410023656 A CN 200410023656A CN 1560226 A CN1560226 A CN 1560226A
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江晓路
管华诗
王静雪
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Ocean University of China
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Abstract

本发明涉及一种新的微生物，特别是涉及一种能产生的琼胶酶的新颖的微生物Alteromonas sp.nov.SY37－12.以及该菌种产生的琼胶酶。本发明的目的是提供一种来源于自然界的新种Alteromonas sp.nov.SY37－12以及利用该菌株生产琼胶酶的方法。本发明从南海海域分离纯化出一株菌Alteromonas sp.nov. SY37－12，其具有产生琼胶酶的特性。该菌种的形态和生理生化特征均不同于已知菌种。根据伯杰氏手册第九版确定其为交替单胞菌属的一个新种。该菌种具有营养要求范围广，容易培养和培养时间短的特点，产生的琼胶酶具有很高的活性。用本发明的菌株作为琼胶酶的生产菌株，具有产品的活性高、稳定性好、生产周期短、成本低的特点，能够实现工业化的生产。该酶作用于琼胶可产生不同分子量大小的琼胶糖片段。

Description

用Alteromonas sp.nov.SY 37-12菌株生产琼胶酶
技术领域
本发明涉及一种新的微生物Alteromonas sp.nov.SY37-12以及该菌株产生的琼胶酶。
背景技术
据本发明人通过查阅资料、文献检索所知，产生琼胶酶的菌种主要来源于海洋。Cytophage，Vibrio；Streptomyces，Alteromonas，Pseudoalteromonas，Pseudomonas中的某些菌株均可以产生琼胶酶。根据伯杰氏手册第九版，本发明的菌株的形态和生理生化特征均不同于已知菌，是从自然界分离出的新种(该菌种区别与其它该属菌的特征见表1)。
发明内容
本发明的目的是提供一种来源于海藻的新种Alteromonas sp.nov.SY37-12以及利用该菌株生产琼胶酶的方法。
本发明分离纯化出一株来源于海藻的新种Alteromonas sp.nov.SY37-12，其具有产生琼胶酶的特性，保藏单位简称CCTCC，保藏号为M204009。该琼胶酶作用于琼胶可产生不同分子量大小的琼胶糖片段。
本发明的特点是：
首次分离出产琼胶酶的Alteromonas sp.nov.SY37-12菌株。图1显示了该菌株的电镜照片。该菌株为杆状，革兰氏阴性菌，0.5～0.6μm×1.8～2.1μm。端生单鞭毛，不积累聚羟丁酸盐颗粒作为胞内贮存物。该菌种来源于海洋环境，生长必须需要NaCl。根据伯杰氏手册第九版确定其为交替单胞菌属。此菌株在2216E培养基上37℃培养24h形成单菌落，菌落乳白色，圆形，边缘整齐。该菌种具有营养要求范围广，容易培养和培养时间短的特点，特别是具有高的产酶活性，在2216E培养基中发酵16h，其发酵液中酶的活力即达到300U/ml。用本发明的菌株作为琼胶酶的生产菌株，具有产品的活性高、稳定性好、生产周期短、成本低的特点，能够实现工业化的生产。
分离纯化后，利用SDS-PAGE测定了该菌株产生的琼胶酶的分子量。图2显示该琼胶酶的分子量为39Kda。由表2可知它不同于已报导的琼胶酶的分子量。
下面通过实施例说明本发明：
实施例
从南海海域采集样品，通过选择菌落在2216E培养基上产生透明圈的大小进行初筛，通过测定酶活力复筛得到具有高产琼胶酶活力的一株细菌。该菌株通过形态观察和生理生化鉴定为交替单胞菌属中的一个新种。(2216E培养基的组成：酵母膏1g，蛋白胨5g，磷酸高铁0.1g，琼胶15g，NaCl 20g，H2O 1000ml)
将菌种用2216E培养基活化后，以1％的接种量接入装有100ml发酵培养基的250ml三角瓶中，35℃摇瓶培养16h后，将培养液用冷冻离心机以4000rpm离心10分钟去除菌体，获得发酵酶液。酶活力测定的条件：1mL酶液加入20mL0.5％琼胶底物中，35℃反应30min。沸水浴灭活5min，用DNS法测定还原糖的量。在上述反应条件下，1mL酶液1min产生1μg还原糖作为一个酶活力单位，以D-半乳糖作为标准。
该菌种产生琼胶酶的分子量测定采用(NH4)2SO4沉淀得到的酶蛋白在50mmol Tris-HCl pH7.5的缓冲液中透析、干燥后。经DEAE-Sepharose离子交换柱层析和Sephacryl S-100凝胶柱层析分离纯化酶蛋白。SDS-PAGE得到一条单带，分子量为39Kda。
                A.aur- A.ci- A.colw- A.denit- A.esp- A.halo-  A.ha- A.luteo-  A.macl- A.nigrif- A.r-  A.un- A.sp.
                antia  trea  elliana rificans ejiana planktis nedai violacea  eodii   aciens    ubra  dina  nov
细胞形状
  直              +      +      +       +        +      +        +      +       +       +        +     -    +
  弯              -      -      -       -        -      -        -      d       -       -        -     +    -
  有鞘鞭毛        -      -              +        -      -        +      d       -       -        -     -    +
  生长4℃         +      -      -       +        -      -        +      d       -       -        -     d    -
  35℃            -      d      -       -        d      d        -      +       +       -        +     -    +
  40℃            -      d      -       -        -      -        -      -       d       -        -     -    +
  硝酸盐还原      -      -      +       -        -      d        +      -       -       -        -     -    -
  反硝化产气      -      -      -       +        -      -        -      -       -       -        -     -    -
  产生物荧光      -      -      -       -        -      -        +      -       -       -        -     -    -
  需有机因子      +      +      +       +        +      d        +      +       -       +        +     +    -
  产生淀粉酶      +      +      +       +        +      d        -      +       +       -        +     d    +
  藻朊酸酶        -                     +        +      -        -              d       -              -    +
  几丁质酶        -      d      -       +        -      d        +      -       -       -        -     +    -
  利用
  D-葡萄糖        +      +      -       +        +      +        d      +       +                +     +    +
  D-甘露糖        +      +      -                d      +        -      d       -       -        +     -    +
  D-半乳糖        -      -      -       -        +      d        d      -       +       +        -     -    +
  D-果糖          +      +      -       -        d      d        -      d       +       +        -     -
  蔗糖            -      -      -       -        +      +        -      -       +       -        -     +    -
  麦芽糖          d      -      -       +        +      +        -      +       +                -     +    -
  纤维二糖        -      -      -                d      -        -      -       +       -        -     -    -
  蜜二糖          -                              +      -        -      -       +       +        -          -
  乳糖            -      -      -       -        +      -        -      -       +       +        -     -    -
  水杨苷          -      -      -                -      -        -      -       +       -        -     -
  D-葡萄糖酸      -      -      +       -        -      -        d      -       +       +        -     -
  盐
  N-乙基葡萄      +             -       -        -      +        +      +       d       -              +
  糖酸铵
  琥珀酸          -      -      -       -        -      +        -      -       -       +        +           -
  富马酸          -      -      +       -        -      +        -      -       -       +        -     +
  DL-乳酸盐       -      -      -       -        -      -        -      d       d       +        -     -
  DL-甘油酸盐     -             -                -      -        +      -       +       -              -
  L-酮戊二酸      -      -      +                -      -        -      -       -       -        -     -
  柠檬酸盐        -      -      -                +      +        -      -       -       +        -     -     -
  乌头酸盐        -                              +      +        -              -       +              -     -
  D-甘露醇        -      -      -       -        +      d        -      -       d       -        -     -     -
  甘油            -      -      -       -        -      -        -      -       +       +        -     -     -
  L-苏氨酸               -      -                +      -        -      +       -       -                    +
  L-酪氨酸        +             -       +        +      +        d      d       +       +              +     +
  L-谷氨酸        -      -              +        -      d        +      -       d       +              -     -
  L-精氨酸        +      -              +        d      d        -      d       d       -              d     -
  腐胺                          -       -        -      -        +              -       -              -     -
  水溶性褐色素    -      -      +       -        -      -        d      -       -       -        -     -     -
  水溶性红色素    -      -      -       +        -      -        -      -       -       -        +     -     -
  水溶性紫色素    -      -      -       -        -      -        -      +       -       -        -     -     -
  水溶性黄色素    -      +      -       -        -      -        -      +       -       -        -     -     -
  水溶性橙色素    +      -      -       -        -      -        -      -       -       -        -     -     -
  水溶性黑色素    -      -      -       -        -      -        -      -       -       +        -     -     -
                          表1交替单胞菌属种间的鉴别
       菌种                  酶系       分子量 KDa
Vibrio sp.Strain JT0107                    72
Alteromonas sp.E-1                         82
Bacillus cereus ASK202                     90
Vibrio sp.PO-3034            A            87.5
                              B            115
                              C            57
Bacillus sp.MK03                           92
Pseudomonas atlantica                      32
Vibrio sp.AP-2                             20
Alteromonus sp.strain C-1.                 52
Pseudomonas sp.PT-5                        31
Pseudomonas sp.strain W7                   59
Alteromonas sp.nov.SY 37-12                39
表2 Alteromonas sp.nov.SY 37-12菌株产酶与其他菌株产酶的比较

Claims (2)

1：对琼胶酶生产菌种Alteromonas sp.nov.SY 37-12实施专利保护。
2：对利用Alteromonas sp.nov.SY 37-12产生的琼胶酶实施专利保护。
CNB2004100236561A 2004-03-02 2004-03-02 用Alteromonas sp.nov.SY 37－12菌株生产琼胶酶 Expired - Fee Related CN1281732C (zh)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100591755C (zh) * 2005-06-23 2010-02-24 国家***第三海洋研究所 一种抗艾滋病病毒ⅰ型的化合物制备方法
CN101451113B (zh) * 2008-11-06 2010-10-06 国家***第二海洋研究所 一种需钠弧菌及采用该菌生产琼胶酶的方法
CN101608166B (zh) * 2008-05-15 2011-03-16 汕头大学 一株黄杆菌菌株及其在产生琼胶酶中的应用
CN101955895B (zh) * 2010-02-09 2012-05-23 浙江工业大学 玫瑰杆菌及其在制备琼胶寡糖中的应用
CN103352016A (zh) * 2013-06-27 2013-10-16 中国海洋大学 利用Alteromonas colwelliana A321发酵浒苔制备生物肥
CN103451119A (zh) * 2012-06-03 2013-12-18 中国海洋大学 一种交替单胞菌及其用它生产凝胶型浒苔多糖降解酶的方法

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100591755C (zh) * 2005-06-23 2010-02-24 国家***第三海洋研究所 一种抗艾滋病病毒ⅰ型的化合物制备方法
CN101608166B (zh) * 2008-05-15 2011-03-16 汕头大学 一株黄杆菌菌株及其在产生琼胶酶中的应用
CN101451113B (zh) * 2008-11-06 2010-10-06 国家***第二海洋研究所 一种需钠弧菌及采用该菌生产琼胶酶的方法
CN101955895B (zh) * 2010-02-09 2012-05-23 浙江工业大学 玫瑰杆菌及其在制备琼胶寡糖中的应用
CN103451119A (zh) * 2012-06-03 2013-12-18 中国海洋大学 一种交替单胞菌及其用它生产凝胶型浒苔多糖降解酶的方法
CN103451119B (zh) * 2012-06-03 2015-06-17 中国海洋大学 一种交替单胞菌及其用它生产凝胶型浒苔多糖降解酶的方法
CN103352016A (zh) * 2013-06-27 2013-10-16 中国海洋大学 利用Alteromonas colwelliana A321发酵浒苔制备生物肥

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