CN113881655A - 一种角蛋白酶及应用 - Google Patents
一种角蛋白酶及应用 Download PDFInfo
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- CN113881655A CN113881655A CN202111282318.XA CN202111282318A CN113881655A CN 113881655 A CN113881655 A CN 113881655A CN 202111282318 A CN202111282318 A CN 202111282318A CN 113881655 A CN113881655 A CN 113881655A
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- Prior art keywords
- keratinase
- gly
- ala
- lbker
- val
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Abstract
本发明属于蛋白酶技术领域，具体涉及一种角蛋白酶，还涉及上述角蛋白酶在水解动物角质原料中的应用。本发明所述及的角蛋白酶LbKer来源于溶杆菌CGMCC No.16072，其耐受温度大于55℃，在pH范围为5.0‑9.0下具有酶活，该角蛋白酶发酵粗酶液12h对动物角蛋白原料降解率均达到60%以上，并且具有序列表SEQ ID NO.1所示的氨基酸序列，具有上述氨基酸序列的角蛋白酶是目前首次报道的一种新型的蛋白序列，与已报道的几角蛋白酶蛋白序列相似度较低。本发明所提供的角蛋白最大的优点是：具有底物谱较宽、对羊毛降解效率高等特性。
Description
技术领域
本发明属于蛋白酶技术领域，具体涉及一中角蛋白酶，还涉及上述角蛋白酶的应用。
背景技术
畜禽废弃物逐年增长，鉴于其难降解性，给环境保护带来了较大的麻烦。养殖污染被列为需重点治理的污染之一，养殖场畜禽固废污染是养殖业的一大难题。对畜禽固废进行无害化处理，资源化利用，防止和消除养殖场畜禽粪便的污染，对于保护生态环境，推动农业可持续发展和增强农产品市场竞争力具有十分重要的意义。
据统计，全世界每年禽畜羽毛等产量达数百万吨。这些废弃物除少量用于羽绒制品和制备氨基酸外，绝大部分未得到合理利用，不仅造成大量的资源浪费，而且造成严重的环境污染，主要是因为这些废弃物的主要成分是难被降解的角蛋白。角蛋白可分为α角蛋白和β角蛋白两种，α角蛋白主要存在于哺乳动物的毛发、羽毛、兽角、指/趾甲、爪子和兽蹄中。更硬的β角蛋白存在于爬行动物的趾甲、壳、鳞、鸟喙、爪子及刚毛中。其粗蛋白含量在80％以上，各种氨基酸总量在70％以上，同时含有较多大量元素、微量元素和未知生长因子，是一种良好的饲料蛋白及肥料来源，对它的开发利用具有重要的应用前景。
利用微生物降解等生物技术途径解决角蛋白分解利用可以解决传统化学方法存在的二次污染和废弃物难处理等问题。但角蛋白存在大量的二硫键、氢键和疏水作用，不容易被普通蛋白酶水解。角蛋白酶(Keratinase)是一种可以特异性降解角蛋白的酶类，目前已发现30 多种微生物能分泌角蛋白酶，国内外角蛋白酶的研究主要集中在真菌中的皮肤癣菌和白假丝酵母(Candida albicans)，放线菌中的链霉菌 (Streptomyces)和高温单胞菌属(Thermonspora)及细菌中的地衣芽孢杆菌(Bacillus licheniformis)和枯草芽孢杆菌(Bacillus subtilis) 等，但大多数存在底物谱较窄、酶活力不足等问题，目前产业化应用较少。
因此，需要针对上述的缺陷，筛选一种性能优良的新型角蛋白酶，以便于解决羽毛废弃物工业化应用这一技术难题。
发明内容
为了解决上述的技术问题，本发明提供了一种具有序列表SEQ ID NO.1所示的氨基酸序列的角蛋白酶，还提供了上述角蛋白酶的应用方法。上述角蛋白酶LbKer来源于溶杆菌Lysobacter sp.，于2018年 07月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号CGMCC NO.16072，是目前首次报道的一种新型的蛋白序列，与已报道的几丁质脱乙酰基酶蛋白序列相似度较低(如附图1所示的系统进化分析)。
上述角蛋白酶LbKer具有序列表SEQ ID NO.1所示的氨基酸序列；
本发明还保护编码SEQ ID NO.1所示角蛋白酶LbKer的基因；
本发明还提供所述角蛋白酶LbKer在水解角质原料(动物角质) 中的应用。
本发明提供的角蛋白酶LbKer具有以下酶学性质：
(1)角蛋白酶LbKer的最佳反应温度为45℃，可耐受温度大于 55℃；
(2)角蛋白酶LbKer的最佳反应pH为7.0，酶活的pH范围为 5.0-9.0；
(3)角蛋白酶LbKer的底物谱较宽，对鸡毛、鸭毛、鹅毛、羊毛、牛毛和蹄角均表现出了较高的催化活性。
本发明的有益效果在于：
与已经报道的角蛋白酶相比，LbKer的底物谱较宽，能够作用于多种羽毛及蹄角，发酵粗酶液12h对各种角蛋白原料的降解率均达到 60％以上，其中鸡毛和蹄角的降解率达到85％以上，对羊毛的降解率达到75％以上，是目前报道的最快羊毛降解酶。
附图说明：
图1系统进化分析图；
图2酶学特性分析图；
其中，(a)为最适温度曲线；(b)为最适pH曲线；(c)为温度稳定性曲线；(d)为pH稳定性曲线；
图3为实施例3中LbKer应用于羊毛降解的图片；
图4为实施例3中LbKer应用于鸡毛降解的图片；
图5为实施例3中LbKer应用于蹄角降解的图片；
图6为溶杆菌Lysobacter sp.全基因组测序结果图谱。
具体实施方式：
为了能使本领域技术人员更好的理解本发明，现结合具体实施方式对本发明进行更进一步的阐述。
实施例1
本发明所述及的角蛋白酶LbKer具有序列表SEQ ID NO.1所示的氨基酸序列，角蛋白酶LbKer来源于溶杆菌Lysobacter sp.，该溶杆菌于2018年07月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
本发明的所提供的角蛋白酶的底物谱：
实施例1中研究了LbKer的作用底物谱，选择鸡毛、鸭毛、鹅毛、羊毛、牛毛和蹄角作为底物，0.1g底物溶液中加入5mL LbKer粗酶液，在搅拌下，让混合物在45℃和pH8.0下反应12小时。然后将样品煮沸1分钟灭酶，采用过滤称量残留不溶物的方式表征其不同底物的降解能力。
结果如表1所示，LbKer对所有被测底物都有较高活性。与其它已经报道角蛋白酶相比，LbKer对羊毛表现出了更高的活性，同时也表现出了更广的底物谱。
表1.LbKer对不同底物的降解率
表1中，降解率的计算方法如下：
A-—底物酶解抽滤烘干后滤纸质量(g)
A0--滤纸最初的质量(g)
m--羊毛的质量1g。
实施例2
关于本发明的角蛋白酶的酶学特性
(1)为了确定酶的最佳反应温度，将酶在不同温度(30-80℃)， pH7.5条件下反应60min，检测酶活性；
(2)为了研究酶的温度稳定性，将酶在不同温度(30-80℃)下预孵育30min，pH7.5，37℃反应60分钟检测酶的剩余酶活力；
(3)为了确定最佳pH值，测定了酶在37℃，不同pH条件下，反应60min的酶活力；
(4)为了研究酶的pH值稳定性，将酶放置于各种pH5-12缓冲液中预孵育过夜，然后在pH7.5，37℃反应60分钟检测剩余酶活力；
以上酶学特性的检测，是以角蛋白为反应底物，10mg角蛋白固体，加入100μl粗酶液，500ul pH＝7.5的Tris-HCl缓冲液，调节水浴锅温度进行反应，反应完检测酶解液280nm处的吸光值。
本发明所采用的酶活单位的定义：在一定反应条件下(如未特别说明则为pH7.5，37℃)每小时降解1微克角蛋白所需要的酶量定义为一个酶活力单位U。
酶活力计算公式为：
A280：酶解液样品的吸光值；
A0：空白的吸光值；
D：稀释倍数；
T：酶促反应时间，h；
K：线性系数。
结果如图2所示，LbKer的最佳反应温度和pH分别为45℃(图 a)和7.0(图b)。热稳定性试验表明，在40℃以下处理30min后， LbKer的酶活性几乎保持100％，而在55℃及以上完全失活(图c)。酶在pH 5.0～9.0之间酶活力稳定(图d)。
实施例3
LbKer的应用1
称取烘干后的羊毛1g加入50mL角蛋白酶LbKer粗酶液37℃反应12h，采用将反应液采用中性滤纸过滤洗涤，将残留物烘干至恒重称量，计算降解率，羊毛降解率达到76.9％。
LbKer的应用2
称取烘干后的鸡毛0.5g加入25mL角蛋白酶LbKer粗酶液37℃反应12h，采用将反应液采用中性滤纸过滤洗涤，将残留物烘干至恒重称量，计算降解率，鸡毛降解率达到84.1％。
LbKer的应用3
称取烘干后的蹄角1g加入50mL角蛋白酶LbKer粗酶液37℃反应12h，采用将反应液采用中性滤纸过滤洗涤，将残留物烘干至恒重称量，计算降解率，蹄角降解率达到93.6％。从动物蹄角的降解效果来看，本发明的角蛋白酶降解效果优异。
从附图3～5中可以看出，本发明所提供的角蛋白酶LbKer粗酶液在降解羊毛、鸡毛、蹄角中效果优异。以上的案例仅仅是本发明所提供的部分降解效果实例，未提及的比如羽毛等角质含量较高的动物产品，本发明的角蛋白酶也具有优异的效果。
附图6为溶杆菌Lysobacter sp.全基因组测序结果，本发明人获得了本发明的角蛋白酶LbKer。
以上所述仅为本发明的较佳实施案例而已，并不用以限制本发明。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<110> 山东理工大学
<120> 一种角蛋白酶及应用
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Gly Ala Leu Arg Tyr Met Asp Gln Pro Ser Arg Asp Gly Arg Ser Ile
370 375 380
Asp Asn Ala Ser Gln Tyr Tyr Asn Gly Ile Asp Val His His Ser Ser
385 390 395 400
Gly Val Tyr Asn Arg Ala Phe Tyr Leu Leu Ala Asn Ser Pro Gly Trp
405 410 415
Asp Thr Arg Lys Ala Phe Glu Val Phe Val Asp Ala Asn Arg Tyr Tyr
420 425 430
Trp Thr Ala Thr Ser Asn Tyr Asn Ser Gly Ala Cys Gly Val Ile Arg
435 440 445
Ser Ala Gln Asn Arg Asn Tyr Ser Ala Ala Asp Val Thr Arg Ala Phe
450 455 460
Ser Thr Val Gly Val Thr Cys Pro Ser Ala Leu
465 470 475
Claims (10)
1.一种角蛋白酶，其特征在于，其氨基酸序列如序列表SEQ ID NO.1所示。
2.如权利要求1所述的角蛋白酶，其特征在于，所述的角蛋白酶耐受温度大于55℃。
3.如权利要求1所述的角蛋白酶，其特征在于，所述的角蛋白酶在pH范围为5.0-9.0下具有酶活。
4.如权利要求1所述的角蛋白酶，其特征在于，所述的角蛋白酶发酵粗酶液12h对动物角蛋白原料降解率均达到60%以上。
5.如权利要求1所述的角蛋白酶，其特征在于，所述的角蛋白酶发酵粗酶液12h对鸡毛和蹄角的降解率达到85%以上。
6.如权利要求1所述的角蛋白酶，其特征在于，所述的角蛋白酶发酵粗酶液12h对羊毛的降解率达到75%以上。
7.如权利要求1所述的角蛋白酶，其特征在于，所述角蛋白酶LbKer来源于溶杆菌CGMCCNo.16072，所述的溶杆菌于2018年07月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
8.如权利要求1所述的角蛋白酶的编码基因。
9.如权利要求1所述的角蛋白酶在水解动物角质原料中的应用。
10.如权利要求1所述的角蛋白酶在水解鸡毛、鸭毛、鹅毛、羊毛、牛毛或蹄角中的应用。
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