CN114958623B - 一株高产纤维素酶的盖姆斯木霉及其应用 - Google Patents
一株高产纤维素酶的盖姆斯木霉及其应用 Download PDFInfo
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Abstract
本发明公开了一株高产纤维素酶的木霉菌株及其应用。本发明菌株为盖姆斯木霉Trichoderma gamsii TC787菌株,其在液态摇瓶发酵条件下的滤纸酶活、内切酶活和β‑葡萄糖苷酶活分别达到2.21、0.41和1.45IU/mL,在固态发酵条件下,TC787在粉碎的水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质。该菌株对上述基物的降解率分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%。盖姆斯木霉TC787菌株适用于液态发酵产纤维素酶和固态发酵降解木质纤维素。
Description
技术领域
本发明涉及真菌资源及其利用领域的一株高产纤维素酶的盖姆斯木霉及其应用。
背景技术
木质纤维素是地球上储量最大的可再生资源,其中纤维素是其主要成分,约占30%,有效降解纤维素使其转化为可利用的产品或实现农田秸秆分解是当前的研究热点。木霉属真菌是重要的纤维素降解菌,其分泌的纤维素酶能够将纤维素分解成还原糖。工业菌株酶活有待提高、市场成本高等问题阻碍了其应用,农业秸秆还田需要高酶活菌株。因此,筛选新的高产纤维素酶菌株具有重要的理论意义和应用前景。
发明内容
为解决生产中纤维素酶成本高和/或酶活低和/或农业秸秆还田需要高酶活菌株等问题,本发明提供一株高产纤维素酶的盖姆斯木霉菌株,以期高效分解纤维素,解决工农业生产中的问题。
为解决上述技术问题,本发明首先提供了一株盖姆斯木霉,所述盖姆斯木霉为盖姆斯木霉(Trichoderma gamsii),其菌株号为TC787,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.22416。以下简称盖姆斯木霉TC787。
本发明还提供了一种用于制备纤维素酶的培养物,所述培养物含有盖姆斯木霉TC787。
上述培养物中,所述培养物是将盖姆斯木霉TC787在微生物培养基中培养得到的物质(发酵产物)。
上述培养物中,所述物质包括所述盖姆斯木霉TC787和盖姆斯木霉TC787的代谢物。
上述培养物中,所述微生物培养基可为固体培养基或液体培养基。
术语“培养物”是指经人工接种和培养后,长有微生物群体的液体或固体产物(发酵产物)的统称。即通过将微生物进行生长和/或扩增而获得的产物,其可以是微生物的生物学纯培养物,也可以含有一定量的培养基、代谢物或培养过程中产生的其他成分。术语“培养物”还包括通过将微生物传代而获得的传代培养物,其可以是某一代的培养物,也可以是若干代的混合物。
上述盖姆斯木霉TC787或其培养物在制备纤维素酶中的应用也是本发明保护的范围。
本发明的另一个目的是提供一种制备纤维素酶的方法。
本发明所提供的制备纤维素酶的方法是利用盖姆斯木霉TC787进行发酵得到纤维素酶。
上述方法中,所述纤维素酶可为内切型葡聚糖酶、外切型葡聚糖酶和/或β-葡萄糖苷酶。
上述方法中,所述发酵包括用培养基培养所述盖姆斯木霉TC787,所述培养基可为液体培养基或固体培养基;所述固体培养基可含有基质粉,所述基质粉可来源于浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片中的至少一种。
上述方法中,所述固体培养基可由固体基物与基础营养盐培养基组成。
所述固体培养基中,所述固体基物与基础营养盐培养基的质量比可为1:2.4-2.6;1L所述基础营养盐培养基的组成可为:(NH4)2SO4 4.9-5.1g/L,KH2PO4 2.9-3.1g/L,MgSO4·7H2O 0.49-0.51g/L,CaCl2 0.49-0.51g/L,其余为水;所述固体基物由所述基质粉与麸皮组成,所述固体基物中,所述基质粉与所述麸皮的质量比可为1:1。
盖姆斯木霉TC787或其培养物在降解纤维素中的应用也是本发明保护的范围。
在上述降解纤维素中的应用中,所述纤维素可来源于下述至少一种:浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片。
实验证明,盖姆斯木霉TC787在液态发酵条件下,滤纸酶活、内切酶活和β-葡萄糖苷酶活力分别达到2.21、0.41和1.45IU/mL发酵液,分别是相同条件下里氏木霉QM9414酶活的2.22倍、0.85倍和3.30倍。表明盖姆斯木霉TC787在实验室条件下液态发酵具有良好的产纤维素酶能力。在固态发酵条件下,盖姆斯木霉TC787在水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下,均能够诱导产纤维素酶,发酵6天,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质(如图3所示)。盖姆斯木霉TC787对上述6种基物的降解率(失重率)分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%(如图4所示)。在固态发酵条件下,盖姆斯木霉TC787表现出良好的基物降解能力和产纤维素酶性能。盖姆斯木霉TC787液态和固态发酵均展现出良好的产纤维素酶潜力,是一株有应用潜力的真菌资源,其产酶性能良好,有工业生产纤维素酶和农作物秸秆降解还田循环利用的优势。
保藏说明
菌种名称:盖姆斯木霉
拉丁名:Trichoderma gamsii
菌株编号:TC787
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2021年04月23日
保藏中心登记入册编号:CGMCC No.22416
附图说明
图1为盖姆斯木霉TC787的形态特征。
a-c.25℃培养7天的菌落形态(a.CMD,b.PDA,c.SNA);d,e.产孢簇(d.CMD,e.SNA);f-l.分生孢子梗和瓶梗;m.厚垣孢子;n,o.分生孢子。标尺:a–c=20mm;d,e=200μm;f-h=20μm;i,j=10μm;k-o=5μm。
图2为液态发酵条件下盖姆斯木霉TC787与里氏木霉QM9414发酵192h产酶能力的对比。
图3为固态发酵条件下盖姆斯木霉TC787对6种植物性基物的降解能力。
图4为固态发酵条件下6种不同基物对盖姆斯木霉TC787产纤维素酶的诱导。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中的定量实验,如无特别说明,实验均设置三次重复。
实施例1、盖姆斯木霉TC787 CGMCC No.22416的分离、纯化、保藏和鉴定
1.盖姆斯木霉TC787 CGMCC No.22416的分离纯化和保存
土壤样品取自黑龙江省大兴安岭南瓮河自然保护区,称1g土壤样品,用无菌水稀释至10-1、10-2和10-3三个浓度,每个浓度取200μl涂布于PDA培养基(马铃薯200g,葡萄糖20g,琼脂20g,蒸馏水定容至1000mL,高压灭菌,添加氯霉素300mg/L)平板表面。在25℃下将平板倒置培养1–3天后,挑取单菌落转移至PDA斜面培养基保存。取编号为TC787的菌株(即盖姆斯木霉TC787)进行下述鉴定。
2.盖姆斯木霉TC787的鉴定
盖姆斯木霉TC787生物学特性(如图1所示):在CMD培养基(玉米粉40g/L,葡萄糖20g/L,琼脂20g/L,其余为水)上,25℃培养3天后菌落半径65-67mm,3天长满平板;菌落近无色,放射状,轮廓清晰,气生菌丝丰富,集中在菌落边缘,羊毛状,延伸到培养皿盖;分生孢子15天后产生于产孢簇内,产孢簇位于气生菌丝上,数量较多,白色,直径<1mm;厚垣孢子常见。在PDA培养基上,25℃培养3天后菌落直径69-70mm,3天长满平板,菌落与CMD培养基上的类似,但气生菌丝更丰富;分生孢子5天后产生于气生菌丝上;厚垣孢子丰富。在SNA培养基(KH2PO4 1g/L,KNO31g/L,MgSO4·7H2O 0.5g/L,KCl 0.5g/L,葡萄糖0.2g/L,蔗糖0.2g/L,琼脂20g/L,其余为水)上,25℃培养3天后菌落半径41-46mm,5天长满平板,菌落与CMD培养基上的类似;分生孢子5天后产生于气生菌丝上,7天后出现产孢簇,产孢簇较少,主要分布在菌落边缘,初白色,8天后变为绿色;分生孢子梗trichoderma型;瓶梗单生,对生或3轮生,窄烧瓶型,少数锥形,8.1-12.4(-14.2)×2.1-3.1μm,长宽比2.9-5.0(-6.0),基部宽1.4-2.5μm;分生孢子绿色,形状多变,球形,近球形,卵圆形或椭球形,表面光滑,具若干滴状斑点,3.3-4.7(-5.6)×2.8-3.6μm,长宽比1.0-1.5;厚垣孢子常见,顶生。该菌株在上述三种培养基上均无明显气味和色素产生。
基于盖姆斯木霉TC787的两个DNA片段(ITS、tef1)的分子生物学鉴定:
(1)采用CTAB法提取真菌基因组;
(2)ITS和tef1基因片段的扩增及测序:
PCR扩增引物和测序引物分别见表1和表2,PCR反应体系和反应程序分别见表3和表4。反应结束后取2μL扩增产物进行1.0%琼脂糖凝胶电泳检测,凝胶成像系统拍照,由测序公司进行双向测序,获得碱基序列。
表1 PCR扩增引物
表2 PCR测序引物
表3 PCR反应体系(25μL)
表4 PCR反应程序
(3)序列分析
利用Clustal X程序和Bioedit等软件对测序结果进编辑和分析。
用引物ITS4、ITS5测序拼接后得到的ITS序列见SEQ ID No.1。
用引物TEF-1和TEF-2测序拼接后得到的tef1序列见SEQ ID No.2。
将获得的序列与GenBank上的进行比对。结合形态学特征、培养特性和DNA序列数据,将TC787菌株鉴定为盖姆斯木霉(Trichoderma gamsii)。
3.盖姆斯木霉TC787菌株的保藏
盖姆斯木霉TC787已于2021年4月23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,保藏号为CGMCC No.22416。
实施例2、盖姆斯木霉TC787和里氏木霉QM9414液体发酵产酶特性研究
1.液体发酵
1)菌株活化:将斜面保存的盖姆斯木霉TC787和里氏木霉QM9414(TRICHODERMAREESEI QM 9414)(Lars G.FAGERSTAM and L.G&an PETTERSSON.THE CELLULOLYTICCOMPLEX OF TRICHODERMA REESEI QM 9414.Volume 98,number 2.FEBSLETTERS.February 1979)在PDA平板上活化,25℃培养3–5d,待其产生大量的分生孢子。向平板中加入5–10mL无菌水,使用1000μL移液枪轻吸打匀。为了避免培养基中残留物的影响,将收集的分生孢子液通过200目过滤筛过滤,收集滤液,4℃,2000g离心,去除上清,备用。吸取少量悬浮液进行稀释(根据悬浮液浓度,稀释10-103倍不等),利用血球计数板进行计数。
2)孢子接种培养:调节孢子悬浮液浓度为5×106cfu/mL(每毫升中的菌落形成单位,即孢子数目),接种至100mL酶诱导培养基中,25℃,180r/min,恒温培养192h,每个处理设置3次重复。其中,酶诱导培养基(1L):微晶纤维素粉10.0g,Protease peptone 1.0g,磷酸氢二钾2.0g,硫酸铵1.4g,七水硫酸镁0.3g,二水氯化钙0.3g,尿素0.3g,七水硫酸亚铁5.0mg,一水硫酸锰1.6mg,七水硫酸锌1.4mg,二水氯化钴2.0mg,加蒸馏水定容,调pH至5.0,115℃,20min高压蒸汽灭菌。待发酵结束后,取2mL发酵液经过12000r/min离心15min后,立即取上清液进行酶活检测。上述实验重复3次,每次重复采用100ml PD培养基(马铃薯200g,葡萄糖20g,蒸馏水定容至1000mL,高压灭菌得到的液体培养基)进行发酵。
2、木质纤维素酶活性测定
1)滤纸酶酶活的测定方法(DNS法):在容积为1.5mL的离心管中,置入50μL pH4.8,50mMol/L(以下简称mM)的柠檬酸缓冲液(一水柠檬酸210g,无菌水750mL,加入NaOH(50-60g),稀释至1000ml,获得pH 4.5的1mol/L柠檬酸缓冲液。当稀释至50mM时,pH值为4.8)和1cm×1cm的Whatman No.1滤纸条。加入适当稀释的酶液25μL,50℃恒温水浴1h。加入150μL DNS溶液(Ghose,1987,Distilled water 1416.0mL,3,5-Dinitrosalicylic acid10.6g,NaOH 19.8g,溶解后加入:Rochelle salts 306.0g,Phenol(melt at 50℃)7.6mL,Na2S2O5 8.3g,配置完成后,棕色瓶保存,静置7天后备用),沸水浴10min,冷水冷却。加入1mLddH2O,OD540 nm下测定光吸收值,测算酶活力。每个处理设置3次重复,灭活酶液作为对照。滤纸酶酶活力按照国际单位定义为:在pH 4.8,50℃下每分钟催化水解生成1μmol葡萄糖的酶量为一个酶活力单位IU。
2)内切酶酶活(羧甲基纤维素酶酶活)的测定方法(CMC糖化力法):在容积为1.5mL的离心管中,pH 4.8,50mM的柠檬酸缓冲液(体积为50μL)(一水柠檬酸210g,无菌水750mL,加入NaOH(50-60g),稀释至1000ml,获得pH 4.5的1mol/L柠檬酸缓冲液。当稀释至50mM时,pH值为4.8),置入2%CMC-Na(羧甲基纤维素钠)溶液150μL和适当稀释的酶液50μL,50℃恒温水浴30min。加入1mol/L NaOH溶液50μL和150μL DNS溶液,沸水浴10min,冷水冷却。加入850μL ddH2O,OD540nm下测定光吸收值,测算酶活力。每个处理设置3次重复,灭活酶液作为对照。内切酶酶活力按照国际单位定义为:在pH 4.8,50℃下,每分钟催化水解生成1μmol葡萄糖的酶量为一个酶活力单位IU。
3)β-葡萄糖苷酶酶活力测定:1.5mL的离心管中,加入适当稀释的酶液50μL和柠檬酸缓冲液250μL(浓度为50mM,pH 4.6),50℃恒温水浴10min。加入250μL,50℃预热10min5mM的pNPG溶液(精确称取对硝基苯基-β-D-吡喃半乳糖苷(pNPG)0.3013g,置于100mL容量瓶中,加50mM柠檬酸缓冲液(制备方法同上,pH 4.6)定容至100mL,获得10mM的pNPG溶液,再将其稀释一倍得到5mM的pNPG溶液),50℃恒温水浴10min。加入250μL 1mol/L Na2CO3溶液终止反应,OD410 nm下测定光吸收值,测算酶活力。对照为失活酶液,每个处理设置3次重复。β-葡萄糖苷酶酶活力的定义:在pH 4.6,50℃下,每分钟催化pNPG水解生成1μmol对硝基苯酚(pNP)的酶量为一个单位(IU)。
经测定(如图2所示)盖姆斯木霉TC787的滤纸酶活、内切酶活和β-葡萄糖苷酶活力分别达到2.21、0.41和1.45IU/mL,分别是相同条件下里氏木霉QM9414酶活的2.22倍、0.85倍和3.30倍。表明盖姆斯木霉TC787在实验室条件下液态发酵具有良好的产纤维素酶能力。
实施例3、盖姆斯木霉TC787在固态发酵条件下产滤纸酶酶活和对基物降解能力的测试
分别将水稻秸秆、玉米秸秆、小麦秸秆、浒苔、悬铃木叶片和羊茅草粉碎后过50目筛,分别得到水稻秸秆粉、玉米秸秆粉、小麦秸秆粉、浒苔粉、悬铃木叶片粉和羊茅草粉这6种基质粉。固体基物酶诱导培养基:由固体基物与基础营养盐培养基按照1:2.5的质量比混合高压灭菌得到的培养基,其中,基础营养盐培养基(1L)的组成为:(NH4)2SO4 0.50g/L,KH2PO4 3.0g/L,MgSO4·7H2O 0.50g/L,CaCl2 0.50g/L,其余为水。其中,固体基物由基质粉(分别为水稻秸秆粉、玉米秸秆粉、小麦秸秆粉、浒苔粉、悬铃木叶片粉和羊茅草粉中的任一种)与麸皮比按照1:1的质量比混合而成。分别得到水稻秸秆粉酶诱导培养基(图3和图4中简称水稻秸秆粉)、玉米秸秆粉酶诱导培养基(图3和图4中简称玉米秸秆粉)、小麦秸秆粉酶诱导培养基(图3和图4中简称小麦秸秆粉)、浒苔粉酶诱导培养基(图3和图4中简称浒苔粉)、悬铃木叶片粉酶诱导培养基(图3和图4中简称悬铃木叶片粉)和羊茅草粉酶诱导培养基(图3和图4中简称羊茅草粉)。
1)菌株活化:将斜面保存的盖姆斯木霉TC787在PDA平板上活化,25℃培养3–5d,待其产生大量的分生孢子。孢子悬液的制备:参试菌株在PDA培养基上恒温25℃培养7d,利用无菌蒸馏水冲洗孢子,移液枪将其转移到10mL灭菌离心管中,震荡摇匀。计数,调整孢子浓度至107cfu/mL,作为种子培养液,备用。
2)固态摇瓶发酵:将种子培养液分别转移至上述固体基物酶诱导培养基(水稻秸秆粉酶诱导培养基、玉米秸秆粉酶诱导培养基、小麦秸秆粉酶诱导培养基、浒苔粉酶诱导培养基、悬铃木叶片粉酶诱导培养基和羊茅草粉酶诱导培养基)中,孢子含量均调整为107cfu/g(每克基质中的菌落形成单位,即孢子数目),接种后,8层纱布封口以保证通气量,以100r/min摇动三角瓶,防止培养基板结影响通气,将培养瓶置于摇床30℃培养,分别得到水稻秸秆粉固体曲、玉米秸秆粉固体曲、小麦秸秆粉固体曲、浒苔粉固体曲、悬铃木叶片粉固体曲和羊茅草粉固体曲6种固体曲。这6种固体曲是培养容器内的所有物质,该培养物含有盖姆斯木霉TC787和盖姆斯木霉TC787的代谢物。
3)粗酶液制备:向上述每一种固体曲中加入蒸馏水50mL,40℃摇床浸提1h,过滤,滤液12000rpm离心15min,去除孢子,取上清液(粗酶液)放置10min后待测。
4)滤纸酶酶活性测定:同实施例2。
5)失重率测定:失重率以质量分数ωx计,数值以百分数表示,按照式(1)计算秸秆失重率:ωx=(N0-NX)/N0*100%,式中:NO—腐解前秸秆的干重,克(g);NX—某腐解时间秸秆的干重,克(g)。3次测算取算数平均值作为结果,结果精确至小数点后两位。
盖姆斯木霉TC787在水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下能够产纤维素酶,发酵6天,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质(如图3所示)。TC787对6种基物的降解率(失重率)分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%(如图4所示)。在固态发酵条件下,盖姆斯木霉TC787表现出良好的基物降解能力和产纤维素酶性能。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 中国科学院微生物研究所
<120> 一株高产纤维素酶的盖姆斯木霉及其应用
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 602
<212> DNA
<213> 盖姆斯木霉(Trichoderma gamsii)
<400> 1
ggggggggac agcggaggga tcattaccga gtttacaact cccaaaccca atgtgaacca 60
taccaaactg ttgcctcggc ggggtcacgc cccgggtgcg tcgcagcccc ggaaccaggc 120
gcccgccgga gggaccaacc aaactctttt ctgtagtccc ctcgcggacg ttatttctta 180
cagctctgag caaaaattca aaatgaatca aaactttcaa caacggatct cttggttctg 240
gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 300
catcgaatct ttgaacgcac attgcgcccg ccagtattct ggcgggcatg cctgtccgag 360
cgtcatttca accctcgaac ccctccgggg ggtcggcgtt ggggatcggg aacccctaag 420
acgggatccc ggccccgaaa tacagtggcg gtctcgccgc agcctctact gcgcagtagt 480
ttgcacaact cgcaccggga gcgcggcgcg tccacgtccg taaaacaccc aacttctgaa 540
atgttgacct cggatcaggt aggaataccc gctgaactta agcatatcaa taagcggagg 600
aa 602
<210> 2
<211> 1364
<212> DNA
<213> 盖姆斯木霉(Trichoderma gamsii)
<400> 2
tcgagaaagt tcgagaaggt aagctaattt cactattttt atcactacgc tttattggca 60
cagtcgtgcg tccgacaatc ctgttctcag tcttgtcaat ttttctctcg catcgtcaca 120
ccccgctcta cctgtctcta cccctccttt ggcacagcaa aaattttctg gctgccttgt 180
ttggttttta gtggggtgcc agcttttttt tttctggcaa ccccgctaat cgccgctgtc 240
cctcatccat cgtcttaaca atttgttcac tcaatcgcat ctcattttct ccgtggttca 300
atgtgctgat catgattcaa tcaataggaa gccgccgaac tcggcaaggg ttctttcaag 360
tatgcgtggg ttcttgacaa gctcaaggcc gagcgtgagc gtggtatcac catcgacatt 420
gccctctgga agttcgagac tcccaagtac tatgtcaccg tcattggtat gttttagtat 480
cctcattggc gtttcgaaat catgattcta acgtgccact ctacagacgc tcccggccac 540
cgtgatttca tcaagaacat gatcactggt acctcccagg ctgactgcgc tatcctgatt 600
atcgctgccg gtactggtga gttcgaggct ggtatctcca aggatggcca gacccgtgag 660
cacgctcttc tcgcctacac cctgggtgtc aagcagctca tcgttgccat caacaagatg 720
gacactgcca actgggctga ggctcgttac cttgagatca tcaaggagac ctccaacttc 780
atcaagaagg tcggcttcaa ccccaagacc gttgccttcg tccccatctc cggcttcaac 840
ggtgacaaca tgttggccgc ctccaccaac tgcccctggt acaagggctg ggagaaggag 900
accaaggctg gcaagtccac cggcaagacc cttctcgagg ccattgacgc cattgagccc 960
cccaagcgtc ccacagacaa gccccttcgt ctgccccttc aggatgttta caagatcggt 1020
ggtatcggaa cagtccctgt cggccgtatc gagactggta tcctcaagcc cggtatggtc 1080
gttaccttcg ctccctccaa cgtcaccact gaagtcaagt ccgttgagat gcaccacgag 1140
cagctcgttg agggtgtccc cggtgacaac gttggtttca acgtcaagaa cgtctccgtc 1200
aaggatatcc gccgtggtaa cgttgccggt gactccaaga acgacccccc catgggtgcc 1260
gcttctttca acgcccaggt catcgtcatg aaccaccctg gccaggtcgg tgccggatac 1320
gctcccgtcc tcgattgcca caccgcccac attgcttgca agtt 1364
Claims (10)
1.盖姆斯木霉,其特征在于:所述盖姆斯木霉为盖姆斯木霉(Trichoderma gamsii)TC787,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCCNo.22416。
2.用于制备纤维素酶的培养物,其特征在于:所述培养物含有权利要求1所述的盖姆斯木霉。
3.如权利要求2所述的培养物,其特征在于:所述培养物是将所述盖姆斯木霉在微生物培养基中培养得到的物质。
4.权利要求1所述的盖姆斯木霉或权利要求2或3所述的培养物在制备纤维素酶中的应用。
5.一种制备纤维素酶的方法,其特征在于:该方法是利用权利要求1所述的盖姆斯木霉进行发酵得到纤维素酶。
6.如权利要求5所述的方法,其特征在于:所述纤维素酶为内切型葡聚糖酶、外切型葡聚糖酶和/或β-葡萄糖苷酶。
7.如权利要求6所述的方法,其特征在于:所述发酵包括用培养基培养所述盖姆斯木霉,所述培养基为液体培养基或固体培养基;所述固体培养基含有基质粉,所述基质粉来源于浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片中的至少一种。
8.如权利要求7所述的方法,其特征在于:所述固体培养基由固体基物与基础营养盐培养基组成,所述固体培养基中,所述固体基物与基础营养盐培养基的质量比为1:2.5;1L所述基础营养盐培养基的组成为:(NH4)2SO44.9-5.1g/L,KH2PO42.9-3.1g/L,MgSO4·7H2O0.49-0.51g/L,CaCl20.49-0.51g/L,其余为水;所述固体基物由所述基质粉与麸皮组成,所述固体基物中,所述基质粉与所述麸皮的质量比为1:1。
9.权利要求1所述的盖姆斯木霉或权利要求2或3所述的培养物在降解纤维素中的应用。
10.如权利要求9所述的应用,其特征在于:所述纤维素来源于下述至少一种:浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片。
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