CN113106044A - 一种链霉菌改造菌及其在降解羽毛中的应用 - Google Patents
一种链霉菌改造菌及其在降解羽毛中的应用 Download PDFInfo
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Abstract
本发明公开了一种链霉菌改造菌及其在降解羽毛中的应用。本发明菌株是半胱氨酸双加氧酶CDO1和蛋白酶Sep39过表达的链霉菌SCUT‑1重组菌。该菌可高效降解羽毛,其固态发酵工艺可将羽毛中难以降解的角蛋白,转化为可溶性蛋白和游离氨基酸,并表现出比野生型链霉菌SCUT‑1更高的降解率和蛋白转化率。以羽毛为唯一的碳源和氮源制成发酵培养基,向发酵培养基中接种本发明的链霉菌改造菌,进行固态发酵,即可实现羽毛的高效降解,可溶性蛋白和氨基酸含量高达0.2g/g和0.29g/g。通过上述方法获得的羽毛粉是非常好的动物、植物和微生物蛋白质营养,可为禽畜类饲料提供更有营养的添加剂或替代品,具有良好的应用前景。
Description
技术领域
本发明涉及基因工程技术领域,特别涉及一种链霉菌改造菌及其在降解羽毛中的应用。
背景技术
农业活动的核心目的是为人类生产有机营养,而含氮营养源的成本最高。作物生产中50%的氮输入来自化肥,另外50%由与植物共生的固氮生物完成。每年生产106吨氮肥消耗了世界1.5%的能源消耗,最终只有约12%的氮通过动植物食品来源流向人类。在所有这些农业活动中,产生了大量的含氮废物,其中羽毛是最丰富的废物之一,全世界每年产生超过千万吨。羽毛主要由β-角蛋白组成,其蛋白质含量约为85%,家禽羽毛约占活家禽重量的5-10%是肥料和动物饲料的宝贵来源。然而,由于含有大量的二硫键,其极难降解且不能被动物消化。
物理和化学法处理羽毛,例如热碱法(Cheong,C.W.,et al.2018Chicken feathervalorization by thermal alkaline pretreatment followed by enzymatichydrolysis for protein-rich hydrolysate production.Waste Management 79:658-666.)和蒸汽爆炸法(专利CN200910170082.3),不仅需要较高的能耗和腐蚀性化学物质且伴随着大量污染物的产生,还会对氨基酸造成一定程度的破坏。
为了开发更绿色更高效的羽毛降解方法,人们对角蛋白酶进行了深入研究,并开发了一些重组角蛋白酶。但在二硫键没有断裂的情况下,角蛋白酶的羽毛水解效率太低,无法满足工业需求(专利CN201911199170.6),且酶的高成本和需在溶液中水解羽毛的特点导致难以实现大批量处理。羽毛降解菌可以直接提供还原力和蛋白酶降解羽毛,被认为是成本更低,更绿色的选择。地衣芽孢杆菌ABTNL-1 36小时在1%的羽毛培养基中降解率可达85%(专利CN201710605993.9)。苏云金芽孢杆菌ABTNL-4水解1%的羽毛培养基,降解率也可达95%(专利CN201710605967.6)。地衣芽孢杆菌BBE11-1和嗜麦芽孢杆菌BBE11-1的共培养法发酵可有效在48小时降解5%的羽毛,降解率可达81.8%,蛋白转化率可达70%(Peng,Z.,et al.2019Effective biodegradation of chicken feather waste by co-cultivation of keratinase producing strains.Microbial cell factories 18(1):84.)(专利CN201711236769.3)。这些羽毛降解菌都需要高含水量的液态发酵(羽毛含量≤5%)才能降解羽毛,这降低了批量处理效率,且烘干步骤增加了用作动物饲料的成本,削弱了工业应用性。在工业生产中,由于固态发酵具有能耗低,耗水少和设备简单的优点,因此被广泛使用。近期我们小组报道了一株高效羽毛降解菌——链霉菌SCUT-1,其不仅可在48小时内完全降解1%的羽毛,还可在40%的羽毛固态发酵中第六天表现出很高的降解率(>50%),其中可溶性肽有0.12g/g,可溶性氨基酸有0.22g/g(专利CN110317748A),但仍有66%不能直接溶解,而优质多肽氨基酸饲料添加剂约为50%,具有进一步改进提升其经济和营养价值的空间。
羽毛降解菌中过表达蛋白酶也是一种有效提升水解效率的方法,在淀粉芽孢杆菌K11中过表达蛋白酶KerK可以提高羽毛水解速度--完全水解羽毛的时间从24小时降低至12小时(Yang,L.,et al.2016Construction of a Rapid Feather-Degrading Bacterium byOverexpression of a Highly Efficient Alkaline Keratinase in Its Parent StrainBacillus amyloliquefaciens K11.Journal of Agricultural and Food Chemistry 64(1):78-84.),但仅在0.4%的羽毛培养基中实现。在羽毛降解菌降解羽毛的过程中,二硫键的断裂被认为是另一个水解羽毛的关键因素,但其机理尚不明确。目前,已经提出了一些可能的机制,包括二硫键还原酶、通过亚硫酸盐的硫解反应和带有巯基的还原性物质(半胱氨酰甘氨酸)(Sharma,R.,and R.Gupta 2012Coupled action of gamma-glutamyltranspeptidase-glutathione and keratinase effectively degrades featherkeratin and surrogate prion protein,Sup 35NM.Bioresource Technology 120:314-7.),但尚未得到充分验证。半胱氨酸双加氧酶(CDO)被发现是皮肤藓菌感染指甲的关键因素,可以催化半胱氨酸的氧化生成亚硫酸盐(Grumbt,M.,et al.2013Keratin degradationby dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump.TheJournal of Investigative Dermatology 133(6):1550-5.)。目前为止,对半胱氨酸双加氧酶的研究仍处于研究阶段,尚未有人研究其在羽毛水解中的作用。
发明内容
申请人在研究链霉菌SCUT-1的羽毛降解过程中检测到亚硫酸盐的产生,并且在分析比较转录组的数据发现羽毛培养基中半胱氨酸双加氧酶基因(cdo1)的表达量相较LB培养基中上调了50倍,另一蛋白酶基因Sep39在羽毛水解过程中也显著上调450倍。从而提出通过基因工程技术过表达蛋白酶Sep39和半胱氨酸双加氧酶CDO1可能是羽毛降解菌SCUT-1羽毛降解的有效提升方法。开发更高效的羽毛水解重组菌株是一种最有希望,最经济的绿色战略。目前为止,尚未有人在羽毛降解菌中通过同时过表达角蛋白酶和CDO1提高羽毛蛋白转化率。
本发明的首要目的在于克服现有技术的缺点与不足,提供一种链霉菌改造菌。
本发明的另一目的在于提供上述链霉菌改造菌的构建方法。
本发明的再一目的在于提供上述链霉菌改造菌在降解羽毛中的应用。
本发明的目的通过下述技术方案实现:
一种链霉菌改造菌,是以链霉菌(Streptomyces sp.)SCUT-1为出发菌,利用基因工程技术构建的过表达半胱氨酸双加氧酶CDO1和蛋白酶Sep39的重组菌。
所述的半胱氨酸双加氧酶CDO1的氨基酸序列为(亦如SEQ ID NO.1所示):
MTSPPESPAVGPRTTDRLAALVDDIRKAVERGLPPDATAHLVGERLAPHLGAPDLLAPEQCEGDAARYRQHLLHAEADGSFSLVSLVWLPGQSTSVHDHVSWCVTGVHRGEEHERRYRLVPASDGAPARLAATEDAVNPVGAVCGFAPPGDIHRVWNGCSHKAVSLHVYGADVSRLGSSVRRVYDLPADH。
所述的蛋白酶Sep39的氨基酸序列为(亦如SEQ ID NO.2所示):
MKRFRIAALLLAAPTALIPAAGTASAAEAATPVVAVQKAEAGQAVKGNYIVTLKSGVEAEDLTEAKDLSPRHVYSEVLNGFAAKLTDGQLKSLQRDSAVLAIEEDQKVTASATQYSATWGLDRIDQRNLPLSGSYTYNRNGAGVTAYIIDTGLDTYHSEFGGRARNVFDAFGGNGQDCNGHGTHVGGTVGGSTHGVAKGVALRGVKVLDCQGSGSYSGIIAGFDWVRQNAVKPAVANASLGGGYSSAVNNAATNLANSGVHLSVAAGNDNQDACNYSPASAPGALSVAASDSGDRKASFSNYGSCTDLYAPGVSITSARMGGGATAMSGTSMASPHVAGVAALYKANYGDASSSTVNSWIVNNSTTYVISGNYSGTPNRLLFKSGL。
上述链霉菌改造菌的构建方法,包括如下步骤:
S1、以链霉菌SCUT-1的基因组DNA为模板进行PCR扩增得到片段cdo1和sep39;将所得片段经NdeI和EcoRI双酶切后,分别与经同样双酶切的载体pSET152进行连接,制得重组质粒pSET152-cdo1和pSET152-sep39;
S2、以重组质粒pSET152-sep39为模板进行PCR扩增得到片段ermE*p-sep39;使用NdeI对重组质粒pSET152-cdo1进行单酶切,与所得片段ermE*p-sep39进行双片段重组,制得重组质粒pSET152-cdo1-sep39;
S3、将重组质粒pSET152-cdo1-sep39转化ET12567/pUZ8002感受态感受态细胞,经含卡那霉素和氯霉素的LB培养基筛选,挑取单克隆进行菌落PCR验证,制得阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39;
S4、将阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39利用接合转移的方法转移入链霉菌SCUT-1,即获得所述的链霉菌改造菌。
优选的,步骤S1中PCR扩增用到的引物及引物序列如下所示:
cdo1-F:5’-TTTACACATATGACTTCCCCGCCCGAATCACC-3’;
cdo1-R:5’-GAGCGAGAATTCTCAGTGGTCGGCGGGCAG-3’;
sep39-F:5’-TTTACACATATGAAGCGTTTCCGGATCGCAGCCC-3’;
sep39-R:5’-GAGCGAGAATTCGATATCTCAGAGGCCGGACTTGAAC-3’;
步骤S2中PCR扩增用到的引物及引物序列如下所示:
sep39-F-2:5’-CCGCCGACCACTGAGCTAGTATGCATGCGAG-3’;
sep39-R-2:5’-ACAGCTATGACATGATTACGTCAGAGGCCGGACTTGAAC-3’。
优选的,步骤S3中,所述的转化的方法为电转化。
优选的,步骤S4中,所述的将阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39利用接合转移的方法转移入链霉菌SCUT-1的具体操作如下所示:
S41、供体菌的制备:
(1)挑取阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39单菌落,接种在含有50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素的LB液体培养基中,35~40℃、150~250rpm,优选37℃、220rpm条件下培养10~16h,得到种子液;
(2)按1~2%接种量将种子液转接到含有50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素的LB液体培养基中,35~40℃,优选37℃培养至OD600达到0.4~0.6;
(3)离心收集菌体,用无抗LB液体培养基洗涤;
(4)离心收集菌体,用无抗LB液体培养基悬浮,备用;
S42、受体菌的制备:
(1)取链霉菌SCUT-1孢子均匀悬浮于水中,得到孢子悬液;
(2)将孢子悬液加入孢子预萌发培养基,37℃,220rpm预萌发培养3h,备用;
S43、将供体菌和受体菌按9~11:1,优选10:1比例混合,离心,涂布于含有0~30mmol/L,优选10mmol/L镁离子浓度的MS平板上,27~33℃,优选30℃培养14~16h;将无菌水、安普霉素、萘啶酮酸按1mL:1mg:1mg比例混合后覆盖整个接合平板,吹干,然后35~40℃,优选37℃继续培养至长出接合子。
优选的,步骤S42中,所述的预萌发培养基每升的组成如下:10g酪蛋白氨基酸,10g酵母提取物,用蒸馏水溶解并定容至1L。
上述链霉菌改造菌在降解羽毛中的应用。
在所述的应用中,以羽毛为唯一的碳源和氮源制成发酵培养基,向所述的发酵培养基中接种所述的链霉菌改造菌,进行发酵,即可实现羽毛降解。
所述羽毛优选自禽畜羽毛;更优选为鸡毛。鸡毛的蛋白含量为80%~90%。
一种利用上述链霉菌改造菌发酵制备发酵羽毛粉的方法,包括如下步骤:
(1)种子液培养:将所述的链霉菌改造菌接种于种子培养基中,培养至OD600达到8~11得到种子液;
(2)发酵:将步骤(1)中得到的种子液转接羽毛发酵培养基中进行发酵,发酵产物烘干,粉碎,即获得发酵羽毛粉。
步骤(1)中所述的种子培养基每升的组成如下:10g蛋白胨、5g酵母提取物、10gNaCl,用蒸馏水溶解并定容至1L。
步骤(1)中所述的培养的条件为35~42℃、150~250rpm培养15~30h;优选为37℃、220rpm培养24h。
步骤(2)中所述的发酵可以是固态发酵或液态发酵;优选为固态发酵;当所述的发酵为固态发酵时,培养基中羽毛的含量优选35~40wt%,发酵的条件优选为35~45℃发酵5~7天;更优选为40℃发酵6天。
步骤(2)中所述的羽毛发酵培养基的组成优选如下:羽毛35~40wt%、氯化钠0.04~0.06wt%、磷酸二氢钾0.03~0.05wt%、磷酸氢二钾0.02~0.04wt%,蒸馏水余量;更优选如下:羽毛40wt%、氯化钠0.05wt%、磷酸二氢钾0.04wt%、磷酸氢二钾0.03wt%,蒸馏水余量。
步骤(2)中所述种子液转接羽毛发酵培养基过程中,接种量优选按种子液与羽毛的体积(mL)质量(g)比为1~2:10计。
上述链霉菌改造菌或在制备发酵羽毛粉的方法在制备禽畜类饲料中的应用,所述的应用是将采用所述的链霉菌改造菌发酵得到的发酵羽毛粉掺入禽畜类饲料中。
本发明相对于现有技术具有如下的优点及效果:
1.本发明通过基因工程技术构建了一株半胱氨酸双加氧酶CDO1和蛋白酶Sep39的过表达菌株SCUT-Ocdo1-sep39。该菌可高效降解羽毛,其固态发酵工艺可将羽毛中难以降解的角蛋白(85%),转化为可溶性蛋白和游离氨基酸,并表现出比野生型链霉菌SCUT-1更高的降解率和蛋白转化率。
2.该菌株兼备链霉菌产菌丝量大,易于在固态基质生长的优势。
3.本发明以羽毛为唯一的碳源和氮源制成发酵培养基,利用菌株SCUT-Ocdo1-sep39进行固态发酵可以得到高蛋白、高氨基酸的发酵产物,可溶性蛋白和游离氨基酸含量可分别高达0.20g/g和0.29g/g。通过该方法使羽毛中的营养成分被充分利用,可将大量废弃羽毛进行重新利用,变废为宝。
4.本发明采用固态发酵的方法,能有效减少废液的产生,减少加工工序,降低能源消耗。
5.本发明方法获得的羽毛粉是非常好的动物、植物和微生物蛋白质营养,可为禽畜类饲料提供更有营养的添加剂或替代品。
附图说明
图1为本发明链霉菌改造菌的羽毛水解效果拍照图;其中,A为未水解的羽毛,B为改造菌SCUT-Ocdo1-sep39水解5%羽毛培养基两天的状态;C为发酵所得发酵羽毛粉。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1半胱氨酸双加氧酶CDO1和蛋白酶Sep39的过表达菌株SCUT-Ocdo1-sep39的构建
(一)链霉菌SCUT-1的培养
将链霉菌SCUT-1(链霉菌(Streptomyces sp.)SCUT-1)接种至高氏1号培养基(可溶性淀粉20g,氯化钠0.5g,硫酸亚铁0.01g,硝酸钾1g,磷酸氢二钾0.5g,硫酸镁0.5g,琼脂15g,蒸馏水1L;pH7.2)37℃培养5-7天,直至产生灰绿色孢子。链霉菌(Streptomyces sp.)SCUT-1于2019年3月20日保藏于广州市先烈中路100号大院59号楼的广东省微生物菌种保藏中心,保藏编号为GDMCC No:60612,已在中国发明专利申请CN201910491700.8中公开。
(二)链霉菌SCUT-1基因组DNA的提取
(1)用接种环刮取灰绿色孢子至带有玻璃珠的种子液培养基(胰蛋白胨10g,氯化钠10g,酵母提取物5g,蒸馏水1L)37℃培养1天。
(2)取1mL菌液使用细菌基因组提取试剂盒(天根生化科技(北京)有限公司)进行基因组DNA提取,具体步骤见该试剂盒说明书。
(三)载体的构建
(1)以链霉菌SCUT-1的基因组DNA为模板,设计引物cdo1-F(5’-TTTACACATATGACTTCCCCGCCCGAATCACC-3’)(下划线的部分表示NdeI酶切位点)和cdo1-R(5’-GAGCGAGAATTCTCAGTGGTCGGCGGGCAG-3’)(下划线的部分表示EcoRI酶切位点),通过PCR方法获得cdo1片段;以链霉菌SCUT-1的基因组DNA为模板,设计引物sep39-F(5’-TTTACACATATGAAGCGTTTCCGGATCGCAGCCC-3’)(下划线的部分表示NdeI酶切位点)和sep39-R(5’-GAGCGAG AATTCGATATCTCAGAGGCCGGACTTGAAC-3’)(下划线的部分表示EcoRI酶切位点),通过PCR方法获得sep39片段。使用NdeI和EcoRI处理纯化回收的两个片段cdo1和sep39,酶切产物纯化回收后在16℃反应12h条件下连接到已用NdeI和EcoRI处理纯化回收的pSET152载体(NTCC典型培养物保藏中心)上,连接反应体系如表1所示:
表1:连接体系
扩增得到的片段cdo1和sep39的核苷酸序列分别如下所示:
半胱氨酸双加氧酶CDO1(SEQ ID NO.3所示):
ATGACTTCCCCGCCCGAATCACCCGCCGTCGGCCCGCGCACGACCGACCGCCTGGCCGCCCTCGTGGACGACATACGCAAGGCCGTGGAACGCGGCCTCCCCCCGGACGCCACCGCGCACCTGGTCGGCGAGAGACTGGCCCCCCACCTCGGGGCCCCCGACCTGCTCGCGCCCGAGCAGTGCGAGGGCGACGCCGCGCGCTACCGCCAGCACCTGCTGCACGCGGAGGCCGACGGCAGCTTCTCCCTCGTCTCCCTCGTCTGGCTGCCGGGGCAGAGCACCTCCGTGCACGACCACGTCTCCTGGTGCGTCACCGGGGTCCACCGGGGCGAGGAGCACGAGCGCCGCTACCGGCTGGTGCCCGCCTCCGACGGCGCACCGGCGCGGCTGGCCGCCACCGAGGACGCGGTGAACCCCGTGGGCGCGGTGTGCGGCTTCGCCCCGCCCGGCGACATCCACCGCGTGTGGAACGGCTGCTCGCACAAGGCCGTGTCCCTCCACGTCTACGGGGCGGACGTCTCCCGGCTGGGCTCCAGCGTCCGCCGGGTGTACGACCTGCCCGCCGACCACTGA。
蛋白酶Sep39(SEQ ID NO.4所示):
ATGAAGCGTTTCCGGATCGCAGCCCTGCTCCTGGCCGCCCCGACGGCCCTGATACCGGCCGCCGGCACGGCGTCCGCCGCCGAGGCCGCCACCCCGGTCGTCGCGGTCCAGAAGGCCGAGGCCGGCCAGGCCGTGAAGGGCAACTACATCGTCACCCTCAAGTCCGGCGTGGAGGCCGAGGACCTGACGGAGGCGAAGGACCTCTCGCCCCGCCACGTCTACTCCGAGGTGCTCAACGGCTTCGCCGCGAAGCTGACGGACGGCCAGCTGAAGTCGCTGCAGCGCGACTCCGCCGTCCTGGCCATCGAGGAGGACCAGAAGGTCACCGCCTCGGCCACCCAGTACAGCGCCACCTGGGGCCTGGACCGGATCGACCAGCGCAACCTGCCGCTGAGCGGCAGCTACACCTACAACCGCAACGGCGCGGGGGTGACGGCCTACATCATCGACACCGGCCTGGACACCTACCACTCCGAGTTCGGCGGCCGCGCCCGCAACGTCTTCGACGCCTTCGGCGGCAACGGCCAGGACTGCAACGGGCACGGCACCCACGTCGGCGGCACCGTCGGCGGCAGCACCCACGGCGTCGCCAAGGGCGTGGCGCTGCGCGGCGTGAAGGTGCTCGACTGCCAGGGCAGCGGCTCCTACTCGGGCATCATCGCCGGCTTCGACTGGGTGCGGCAGAACGCGGTGAAGCCCGCGGTGGCCAACGCCTCACTGGGCGGCGGCTACTCCTCCGCGGTGAACAACGCGGCCACCAACCTGGCCAACTCCGGCGTGCACCTGTCCGTCGCGGCCGGCAACGACAACCAGGACGCCTGCAACTACTCCCCGGCGAGCGCCCCGGGCGCCCTGAGCGTGGCCGCCTCCGACAGCGGCGACCGCAAGGCGTCGTTCAGCAACTACGGCAGCTGCACGGACCTCTACGCCCCCGGCGTGTCCATCACCTCCGCCCGCATGGGCGGCGGCGCCACCGCGATGAGCGGCACCTCGATGGCCTCCCCGCACGTCGCCGGTGTCGCCGCGCTGTACAAGGCGAACTACGGCGACGCCTCCTCCTCGACCGTCAACAGCTGGATCGTCAACAACTCCACCACCTACGTGATCAGCGGCAACTACAGCGGCACGCCCAACCGCCTGCTGTTCAAGTCCGGCCTCTGA。
将连接产物转化至DH5α中,在含有安普霉素的平板上挑选单菌落,提取质粒进行测序鉴定。将得到的重组载体命名为pSET152-cdo1和pSET152-sep39;
(2)以构建好的质粒pSET152-sep39为模板,设计引物sep39-F-2(5’-CCGCCGACCACTGAGCTAGTATGCATGCGAG-3’)和sep39-R-2(5’-ACAGCTATGACATGATTACGTCAGAGGCCGGACTTGAAC-3’)通过PCR方法获得片段ermE*p-sep39。使用NdeI对pSET152-cdo1进行单酶切,纯化回收后与片段ermE*p-sep39进行双片段重组;连接体系如表2所示:
表2:连接体系
将连接产物转化至DH5α中,在含有安普霉素的平板上挑选单菌落,提取质粒进行测序鉴定。将得到的重组载体命名为pSET152-cdo1-sep39。
(四)ET12567/pUZ8002感受态的制备
(1)活化大肠杆菌ET12567/pUZ8002在LB固体培养基,37℃培养过夜(10~16h);
(2)挑取单菌落,接种在含有10ml的LB液体培养基的50mL锥形瓶中(50μg/mL卡那霉素、25μg/mL氯霉素)中,37℃,220rpm过夜(10~16h)培养,至OD600达到3左右;
(3)按5%接种量将种子液转接到含有100mL LB液体培养基的500mL锥形瓶中,37℃,220rpm培养。2h后每半小时取样检测一次菌液的OD600,至OD600达到0.5~0.8;
(4)将锥形瓶从摇床取出后立即冰浴30min;
(5)收集菌液于50mL离心管中,在3500×g和4℃下离心5~10min,弃上清;
(6)用40mL冰水轻轻重悬菌体,在3500×g和4℃下离心5min,弃上清,重复2次;
(7)用20mL预冷的10%甘油轻轻重悬菌体,在3500rpm和4℃下离心5min,弃上清;
(8)用2mL预冷的10%甘油轻轻重悬菌体,分装到无菌的1.5mL离心管中,每管40μL,-80℃保存。
(五)ET12567/pUZ8002的电转化
(1)将保存在-80℃的感受态细胞冰浴融化,取40μL感受态于1.5mL离心管加入0.5μL质粒pSET152-cdo1-sep39,冰浴5min;
(2)将混合液加入0.2cm间距的电击杯中,缓慢加入不能有气泡;
(3)将电击杯表面擦干,放入电转仪滑块中,2.5kV电击,电击时间在4.5-5.8ms之间为宜;
(4)电击完毕后迅速向电击杯内加入1mL无抗LB液体培养基,用枪头吹吸均匀,转移到1.5mL离心管中,37℃,220rpm复壮1h;
(5)将菌液5500×g离心2min,弃上清后用100μL重悬,涂布于LB平板(50μg/ml安普霉素、50μg/ml卡那霉素、25μg/ml氯霉素),37℃倒置培养12-16h;
(6)挑取单克隆用通用引物M13F/R进行菌落PCR验证。菌落PCR反应体系如表3所示:
表3:菌落PCR体系
通过菌落PCR验证得到的阳性菌落命名为ET12567/pUZ8002/pSET152-cdo1-sep39。
(六)链霉菌SCUT-1和大肠杆菌ET12567/pUZ8002/pSET152-cdo1-sep39接合转移
供体菌的制备:
(1)挑取带有整合质粒pSET152-cdo1-sep39的ET12567/pUZ8002单菌落,接种在含有10mL的LB液体培养基(50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素)的50mL锥形瓶中中,37℃,220rpm过夜(10~16h)培养;
(2)按1~2%接种量将种子液转接到含有100mL LB液体培养基(50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素)的500mL锥形瓶中,37℃培养至OD600达到0.4~0.6;
(3)5000×g,5min离心收集菌体,并用50mL无抗LB洗涤菌体两次;
(4)5000×g,5min离心收集菌体,用10mL无抗LB悬浮备用。
受体菌的制备:
(1)孢子的获取:用牙签轻轻取培养一周以上的固体培养基上的链霉菌SCUT-1孢子,置于水中。加入灭菌的玻璃珠,用摇床或震荡仪震荡,直至孢子均匀悬于水中。15%甘油,-20℃保存;
(2)若是冻存的孢子需先热激孢子:将1mL孢子悬液与2mL TES混合,在50℃热激孢子10min。
(3)冷却至室温后加入3mL 2×孢子预萌发培养基(10g酪蛋白氨基酸,10g酵母提取物,1L蒸馏水),37℃,220rpm分别预萌发培养3h;按照10:1将供体菌和受体菌混合(受体菌数量为107),5000rpm离心2min,涂布于含有10mM镁离子浓度的MS平板上,30℃培养14~16h。在1mL无菌水中加入1mg安普霉素和1mg萘啶酮酸,将其覆盖整个接合平板,在超净台内吹干。37℃培养数天至长出接合子,挑取接合子转移到含50μg/mL安普霉素和50μg/mL萘啶酮酸的高氏一号平板上,并摇菌提基因组,进一步通过PCR使用通用引物M13F/R验证,得到的阳性菌落命名为SCUT-Ocdo1-sep39。
实施例2重组链霉菌SCUT-Ocdo1-sep39羽毛降解能力评估
参考实施例1中的方法,构建半胱氨酸双加氧酶CDO1过表达菌株SCUT-cdo1和蛋白酶Sep39过表达菌株SCUT-sep39。
(1)在灭菌的种子液培养基(蛋白胨10g,酵母提取物5g,NaCl 10g,蒸馏水1L)中加入少量实例1链霉菌SCUT-Ocdo1-sep39孢子,37℃,220rpm培养24h左右,至OD600达到8~10,获得种子液,;
(2)将种子液按1%接种量转接5%羽毛发酵培养基(鸡毛5g,蒸馏水100mL,氯化钠0.05g,磷酸二氢钾0.04g,磷酸氢二钾0.03g)中,40℃发酵2天。结果如图1所示。
(3)每天取2mL发酵液,8000×g离心5min,使用亚硫酸盐检测试剂盒(R-Biopharm)测定发酵液中的亚硫酸盐的含量。
经检测野生型菌株链霉菌SCUT-1的5%羽毛发酵液中亚硫酸盐浓度第一天为26.8mg/L,第二天为38.5mg/L。而重组菌株链霉菌SCUT-Ocdo1-sep39的5%羽毛发酵液中亚硫酸盐浓度第一天提高至46.0mg/L,第二天提高至51.4mg/L。此外,重组菌株链霉菌SCUT-cdo1的5%羽毛发酵液中亚硫酸盐浓度第一天为51.15mg/L,第二天为58.86mg/L。重组菌株链霉菌SCUT-sep39的5%羽毛发酵液中亚硫酸盐浓度第一天为24.88mg/L,第二天为37.78mg/L。
(4)采用福林酚法测角蛋白酶活。称取酪氨酸粉末溶于水制备成0、20、40、60、80和100μg/mL的酪氨酸标准溶液。在1.5mL的离心管中加入100μL不同浓度酪氨酸标准溶液和500μL 0.4M Na2CO3,混匀后加入100μL福林酚试剂。将离心管置于40℃水浴20min,反应结束后取200μL反应液在OD660处测量吸光值。以酪氨酸浓度为横坐标,OD660处吸光值为纵坐标绘制标准曲线。得到的标准曲线公式为:y=0.0143+0.00529x,R2=0.9993。
在1.5mL的离心管中加入100μL待测样品和100μL 2%角蛋白,混匀后将离心管置于50℃中水浴20min,反应结束后加入200μL 0.4M TCA终止反应。14000rpm离心2min后弃沉淀,取100μL上清液于新的1.5mL的离心管中,再加入500μL 0.4M Na2CO3和100μL福林酚试剂,混匀将离心管置于40℃中水浴20min,反应结束后取200μL反应液在OD660处测量吸光值。空白对照为0时间的反应液。根据酪氨酸标准曲线计算角蛋白酶酶活性,以在40℃下每分钟水解酪蛋白产生1μg酪氨酸所需要的酶量为1个蛋白酶活力单位。
测得在野生型菌株链霉菌SCUT-1的5%羽毛发酵液中角蛋白酶酶活第一天为60.9U/mL,第二天为70.7U/mL。而重组菌株链霉菌SCUT-Ocdo1-sep39的5%羽毛发酵液中角蛋白酶酶活第一天提高至76.6U/mL,第二天提高至118.6U/mL。此外,重组菌株链霉菌SCUT-cdo1的5%羽毛发酵液中角蛋白酶酶活第一天为52.08U/mL,第二天为55.70U/mL。重组菌株链霉菌SCUT-sep39的5%羽毛发酵液中角蛋白酶酶活第一天为74.73U/mL,第二天为118.48U/mL。
(5)采用茚三酮法测定水解产物中氨基酸含量。称取异亮氨酸粉末溶于水制备成0、25、50、75、100、125、150、175、200、225、250、275、300、325和350μg/mL的异亮氨酸标准溶液。在1.5mL的离心管中加入200μL的标准溶液和50μL PB Buffer,混匀后再加入50μL茚三酮溶液。将离心管置于90℃水浴锅中水浴30min,随后冷却至室温。再向离心管中加入950μL蒸馏水,混匀后静置5min。在OD570处测量吸光值,以异亮氨酸标准溶液的浓度作为横坐标,吸光值作为纵坐标绘制标准曲线。得到的标准曲线公式为:y=0.0067x-0.9642,R2=0.9944。待测样品的氨基酸含量测定与标准品测定方法相同。
测得在野生型菌株链霉菌SCUT-1的5%羽毛发酵液中氨基酸含量第一天为4.1mg/mL,第二天为6.6mg/mL。而重组菌株链霉菌SCUT-Ocdo1-sep39的5%羽毛发酵液中氨基酸含量第一天提高至5.3mg/mL,第二天提高至9.3mg/mL。此外,重组菌株链霉菌SCUT-cdo1的5%羽毛发酵液中氨基酸含量第一天为4.12mg/mL,第二天为6.7mg/mL。重组菌株链霉菌SCUT-sep39的5%羽毛发酵液中氨基酸含量第一天为5.02mg/mL,第二天为8.57mg/mL。
(6)采用BCA蛋白浓度测定试剂盒(TaKaRa)测定浸提液中的可溶性蛋白含量。
测得在野生型菌株链霉菌SCUT-1的5%羽毛发酵液中可溶性蛋白含量第一天为3.3mg/mL,第二天为4.5mg/mL。而重组菌株链霉菌SCUT-Ocdo1-sep39的5%羽毛发酵液中可溶性蛋白含量第一天提高至4.3mg/mL,第二天提高至6.6mg/mL。此外,重组菌株链霉菌SCUT-cdo1的5%羽毛发酵液中可溶性蛋白含量第一天为3.09mg/mL,第二天为4.47mg/mL。重组菌株链霉菌SCUT-sep39的5%羽毛发酵液中可溶性蛋白含量第一天为3.68mg/mL,第二天为5.9mg/mL。
实施例3重组链霉菌SCUT-Ocdo1-sep39的固态发酵
将种子液按其与羽毛的体积(mL)质量(g)比为10:1的比例转接40%羽毛发酵培养基(鸡毛10g,蒸馏水25mL,氯化钠0.0125g,磷酸二氢钾0.01g,磷酸氢二钾0.0075g)中进行固态发酵,40℃发酵6天。
在羽毛发酵培养基中加入75mL的蒸馏水,于摇床中220rpm充分浸提1小时,过滤后进行可溶性蛋白和氨基酸含量测定。
经检测使用菌株SCUT-Ocdo1-sep39进行羽毛固态发酵第六天得到的可溶性蛋白和氨基酸含量高达0.2g/g和0.29g/g,相较野生型菌株SCUT-1进行羽毛固态发酵得到可溶性蛋白和氨基酸含量仅为0.12g/g和0.22g/g。
实施例4发酵羽毛粉的制备
将种子液按其与羽毛的体积(mL)质量(g)比为10:1的比例转接40%羽毛发酵培养基中进行固态发酵,40℃发酵6天,烘干,粉碎,得到发酵羽毛粉。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 一种链霉菌改造菌及其在降解羽毛中的应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 190
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223>半胱氨酸双加氧酶CDO1的氨基酸序列
<400> 1
Met Thr Ser Pro Pro Glu Ser Pro Ala Val Gly Pro Arg Thr Thr Asp
1 5 10 15
Arg Leu Ala Ala Leu Val Asp Asp Ile Arg Lys Ala Val Glu Arg Gly
20 25 30
Leu Pro Pro Asp Ala Thr Ala His Leu Val Gly Glu Arg Leu Ala Pro
35 40 45
His Leu Gly Ala Pro Asp Leu Leu Ala Pro Glu Gln Cys Glu Gly Asp
50 55 60
Ala Ala Arg Tyr Arg Gln His Leu Leu His Ala Glu Ala Asp Gly Ser
65 70 75 80
Phe Ser Leu Val Ser Leu Val Trp Leu Pro Gly Gln Ser Thr Ser Val
85 90 95
His Asp His Val Ser Trp Cys Val Thr Gly Val His Arg Gly Glu Glu
100 105 110
His Glu Arg Arg Tyr Arg Leu Val Pro Ala Ser Asp Gly Ala Pro Ala
115 120 125
Arg Leu Ala Ala Thr Glu Asp Ala Val Asn Pro Val Gly Ala Val Cys
130 135 140
Gly Phe Ala Pro Pro Gly Asp Ile His Arg Val Trp Asn Gly Cys Ser
145 150 155 160
His Lys Ala Val Ser Leu His Val Tyr Gly Ala Asp Val Ser Arg Leu
165 170 175
Gly Ser Ser Val Arg Arg Val Tyr Asp Leu Pro Ala Asp His
180 185 190
<210> 2
<211> 386
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223>蛋白酶Sep39的氨基酸序列
<400> 2
Met Lys Arg Phe Arg Ile Ala Ala Leu Leu Leu Ala Ala Pro Thr Ala
1 5 10 15
Leu Ile Pro Ala Ala Gly Thr Ala Ser Ala Ala Glu Ala Ala Thr Pro
20 25 30
Val Val Ala Val Gln Lys Ala Glu Ala Gly Gln Ala Val Lys Gly Asn
35 40 45
Tyr Ile Val Thr Leu Lys Ser Gly Val Glu Ala Glu Asp Leu Thr Glu
50 55 60
Ala Lys Asp Leu Ser Pro Arg His Val Tyr Ser Glu Val Leu Asn Gly
65 70 75 80
Phe Ala Ala Lys Leu Thr Asp Gly Gln Leu Lys Ser Leu Gln Arg Asp
85 90 95
Ser Ala Val Leu Ala Ile Glu Glu Asp Gln Lys Val Thr Ala Ser Ala
100 105 110
Thr Gln Tyr Ser Ala Thr Trp Gly Leu Asp Arg Ile Asp Gln Arg Asn
115 120 125
Leu Pro Leu Ser Gly Ser Tyr Thr Tyr Asn Arg Asn Gly Ala Gly Val
130 135 140
Thr Ala Tyr Ile Ile Asp Thr Gly Leu Asp Thr Tyr His Ser Glu Phe
145 150 155 160
Gly Gly Arg Ala Arg Asn Val Phe Asp Ala Phe Gly Gly Asn Gly Gln
165 170 175
Asp Cys Asn Gly His Gly Thr His Val Gly Gly Thr Val Gly Gly Ser
180 185 190
Thr His Gly Val Ala Lys Gly Val Ala Leu Arg Gly Val Lys Val Leu
195 200 205
Asp Cys Gln Gly Ser Gly Ser Tyr Ser Gly Ile Ile Ala Gly Phe Asp
210 215 220
Trp Val Arg Gln Asn Ala Val Lys Pro Ala Val Ala Asn Ala Ser Leu
225 230 235 240
Gly Gly Gly Tyr Ser Ser Ala Val Asn Asn Ala Ala Thr Asn Leu Ala
245 250 255
Asn Ser Gly Val His Leu Ser Val Ala Ala Gly Asn Asp Asn Gln Asp
260 265 270
Ala Cys Asn Tyr Ser Pro Ala Ser Ala Pro Gly Ala Leu Ser Val Ala
275 280 285
Ala Ser Asp Ser Gly Asp Arg Lys Ala Ser Phe Ser Asn Tyr Gly Ser
290 295 300
Cys Thr Asp Leu Tyr Ala Pro Gly Val Ser Ile Thr Ser Ala Arg Met
305 310 315 320
Gly Gly Gly Ala Thr Ala Met Ser Gly Thr Ser Met Ala Ser Pro His
325 330 335
Val Ala Gly Val Ala Ala Leu Tyr Lys Ala Asn Tyr Gly Asp Ala Ser
340 345 350
Ser Ser Thr Val Asn Ser Trp Ile Val Asn Asn Ser Thr Thr Tyr Val
355 360 365
Ile Ser Gly Asn Tyr Ser Gly Thr Pro Asn Arg Leu Leu Phe Lys Ser
370 375 380
Gly Leu
385
<210> 3
<211> 573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223>半胱氨酸双加氧酶CDO1的核苷酸序列
<400> 3
atgacttccc cgcccgaatc acccgccgtc ggcccgcgca cgaccgaccg cctggccgcc 60
ctcgtggacg acatacgcaa ggccgtggaa cgcggcctcc ccccggacgc caccgcgcac 120
ctggtcggcg agagactggc cccccacctc ggggcccccg acctgctcgc gcccgagcag 180
tgcgagggcg acgccgcgcg ctaccgccag cacctgctgc acgcggaggc cgacggcagc 240
ttctccctcg tctccctcgt ctggctgccg gggcagagca cctccgtgca cgaccacgtc 300
tcctggtgcg tcaccggggt ccaccggggc gaggagcacg agcgccgcta ccggctggtg 360
cccgcctccg acggcgcacc ggcgcggctg gccgccaccg aggacgcggt gaaccccgtg 420
ggcgcggtgt gcggcttcgc cccgcccggc gacatccacc gcgtgtggaa cggctgctcg 480
cacaaggccg tgtccctcca cgtctacggg gcggacgtct cccggctggg ctccagcgtc 540
cgccgggtgt acgacctgcc cgccgaccac tga 573
<210> 4
<211> 1161
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223>蛋白酶Sep39的核苷酸序列
<400> 4
atgaagcgtt tccggatcgc agccctgctc ctggccgccc cgacggccct gataccggcc 60
gccggcacgg cgtccgccgc cgaggccgcc accccggtcg tcgcggtcca gaaggccgag 120
gccggccagg ccgtgaaggg caactacatc gtcaccctca agtccggcgt ggaggccgag 180
gacctgacgg aggcgaagga cctctcgccc cgccacgtct actccgaggt gctcaacggc 240
ttcgccgcga agctgacgga cggccagctg aagtcgctgc agcgcgactc cgccgtcctg 300
gccatcgagg aggaccagaa ggtcaccgcc tcggccaccc agtacagcgc cacctggggc 360
ctggaccgga tcgaccagcg caacctgccg ctgagcggca gctacaccta caaccgcaac 420
ggcgcggggg tgacggccta catcatcgac accggcctgg acacctacca ctccgagttc 480
ggcggccgcg cccgcaacgt cttcgacgcc ttcggcggca acggccagga ctgcaacggg 540
cacggcaccc acgtcggcgg caccgtcggc ggcagcaccc acggcgtcgc caagggcgtg 600
gcgctgcgcg gcgtgaaggt gctcgactgc cagggcagcg gctcctactc gggcatcatc 660
gccggcttcg actgggtgcg gcagaacgcg gtgaagcccg cggtggccaa cgcctcactg 720
ggcggcggct actcctccgc ggtgaacaac gcggccacca acctggccaa ctccggcgtg 780
cacctgtccg tcgcggccgg caacgacaac caggacgcct gcaactactc cccggcgagc 840
gccccgggcg ccctgagcgt ggccgcctcc gacagcggcg accgcaaggc gtcgttcagc 900
aactacggca gctgcacgga cctctacgcc cccggcgtgt ccatcacctc cgcccgcatg 960
ggcggcggcg ccaccgcgat gagcggcacc tcgatggcct ccccgcacgt cgccggtgtc 1020
gccgcgctgt acaaggcgaa ctacggcgac gcctcctcct cgaccgtcaa cagctggatc 1080
gtcaacaact ccaccaccta cgtgatcagc ggcaactaca gcggcacgcc caaccgcctg 1140
ctgttcaagt ccggcctctg a 1161
<210> 5
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> cdo1-F
<400> 5
tttacacata tgacttcccc gcccgaatca cc 32
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> cdo1-R
<400> 6
gagcgagaat tctcagtggt cggcgggcag 30
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sep39-F
<400> 7
tttacacata tgaagcgttt ccggatcgca gccc 34
<210> 8
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sep39-R
<400> 8
gagcgagaat tcgatatctc agaggccgga cttgaac 37
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sep39-F-2
<400> 9
ccgccgacca ctgagctagt atgcatgcga g 31
<210> 10
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sep39-R-2
<400> 10
acagctatga catgattacg tcagaggccg gacttgaac 39
Claims (10)
1.一种链霉菌改造菌,其特征在于:
是以链霉菌(Streptomyces sp.)SCUT-1为出发菌,利用基因工程技术构建的过表达半胱氨酸双加氧酶CDO1和蛋白酶Sep39的重组菌。
2.根据权利要求1所述的链霉菌改造菌,其特征在于:
所述的半胱氨酸双加氧酶CDO1的氨基酸序列如SEQ ID NO.1所示;所述的蛋白酶Sep39的氨基酸序列如SEQ ID NO.2所示。
3.权利要求1或2所述的链霉菌改造菌的构建方法,其特征在于:包括如下步骤:
S1、以链霉菌SCUT-1的基因组DNA为模板进行PCR扩增得到片段cdo1和sep39;将所得片段经NdeI和EcoRI双酶切后,分别与经同样双酶切的载体pSET152进行连接,制得重组质粒pSET152-cdo1和pSET152-sep39;
S2、以重组质粒pSET152-sep39为模板进行PCR扩增得到片段ermE*p-sep39;使用NdeI对重组质粒pSET152-cdo1进行单酶切,与所得片段ermE*p-sep39进行双片段重组,制得重组质粒pSET152-cdo1-sep39;
S3、将重组质粒pSET152-cdo1-sep39转化ET12567/pUZ8002感受态感受态细胞,经含卡那霉素和氯霉素的LB培养基筛选,挑取单克隆进行菌落PCR验证,制得阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39;
S4、将阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39利用接合转移的方法转移入链霉菌SCUT-1,即获得所述的链霉菌改造菌。
4.根据权利要求3所述的链霉菌改造菌的构建方法,其特征在于:
步骤S1中PCR扩增用到的引物及引物序列如下所示:
cdo1-F:5’-TTTACACATATGACTTCCCCGCCCGAATCACC-3’;
cdo1-R:5’-GAGCGAGAATTCTCAGTGGTCGGCGGGCAG-3’;
sep39-F:5’-TTTACACATATGAAGCGTTTCCGGATCGCAGCCC-3’;
sep39-R:5’-GAGCGAGAATTCGATATCTCAGAGGCCGGACTTGAAC-3’;
步骤S2中PCR扩增用到的引物及引物序列如下所示:
sep39-F-2:5’-CCGCCGACCACTGAGCTAGTATGCATGCGAG-3’;
sep39-R-2:5’-ACAGCTATGACATGATTACGTCAGAGGCCGGACTTGAAC-3’。
5.根据权利要求3所述的链霉菌改造菌的构建方法,其特征在于:
步骤S3中,所述的转化的方法为电转化;
步骤S4中,所述的将阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39利用接合转移的方法转移入链霉菌SCUT-1的具体操作如下所示:
S41、供体菌的制备:
(1)挑取阳性菌落ET12567/pUZ8002/pSET152-cdo1-sep39单菌落,接种在含有50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素的LB液体培养基中,35~40℃、150~250rpm条件下培养10~16h,得到种子液;
(2)按1~2%接种量将种子液转接到含有50μg/mL卡那霉素、25μg/mL氯霉素、50μg/mL安普霉素的LB液体培养基中,35~40℃培养至OD600达到0.4~0.6;
(3)离心收集菌体,用无抗LB液体培养基洗涤;
(4)离心收集菌体,用无抗LB液体培养基悬浮,备用;
S42、受体菌的制备:
(1)取链霉菌SCUT-1孢子均匀悬浮于水中,得到孢子悬液;
(2)将孢子悬液加入孢子预萌发培养基,37℃,220rpm预萌发培养3h,备用;
S43、将供体菌和受体菌按9~11:1比例混合,离心,涂布于含有0~30mmol/L镁离子浓度的MS平板上,27~33℃培养14~16h;将无菌水、安普霉素、萘啶酮酸按1mL:1mg:1mg比例混合后覆盖整个接合平板,吹干,然后35~40℃继续培养至长出接合子;
步骤S42中,所述的预萌发培养基每升的组成如下:10g酪蛋白氨基酸,10g酵母提取物,用蒸馏水溶解并定容至1L。
6.权利要求1或2所述的链霉菌改造菌在降解羽毛中的应用,其特征在于:
在所述的应用中,以羽毛为唯一的碳源和氮源制成发酵培养基,向所述的发酵培养基中接种所述的链霉菌改造菌,进行发酵,即可实现羽毛降解。
7.根据权利要求6所述的链霉菌改造菌在降解羽毛中的应用,其特征在于:
所述羽毛选自禽畜羽毛;进一步为鸡毛。
8.一种利用权利要求1或2所述的链霉菌改造菌发酵制备发酵羽毛粉的方法,其特征在于:包括如下步骤:
(1)种子液培养:将所述的链霉菌改造菌接种于种子培养基中,培养至OD600达到8~10,得到种子液;
(2)发酵:将步骤(1)中得到的种子液转接羽毛发酵培养基中进行发酵,发酵产物烘干,粉碎,即获得发酵羽毛粉。
9.根据权利要求8所述的方法,其特征在于:
步骤(1)中所述的培养的条件为35~42℃、150~250rpm培养15~30h;
步骤(2)中所述的发酵是固态发酵或液态发酵;当所述的发酵为固态发酵时,培养基中羽毛的含量为35~40wt%,发酵的条件为35~45℃发酵5~7天;进一步为40℃发酵6天;
步骤(2)中所述种子液转接羽毛发酵培养基过程中,接种量按种子液与羽毛的体积(mL)质量(g)比为1~2:10计。
10.权利要求1-2任一项所述的链霉菌改造菌,或权利要求8所述的方法在制备禽畜类饲料中的应用。
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CN110317748A (zh) * | 2019-06-06 | 2019-10-11 | 华南理工大学 | 一株链霉菌菌株及其在降解羽毛中的应用 |
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CN115927332A (zh) * | 2022-10-21 | 2023-04-07 | 华南理工大学 | 一种过表达蛋白酶的启动子、链霉菌重组菌及其构建方法与应用 |
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CN115927332B (zh) * | 2022-10-21 | 2023-09-26 | 华南理工大学 | 一种过表达蛋白酶的启动子、链霉菌重组菌及其构建方法与应用 |
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