CN103131681B - 深绿木霉菌株b8-1-34在制备漆酶方面的应用 - Google Patents
深绿木霉菌株b8-1-34在制备漆酶方面的应用 Download PDFInfo
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Abstract
本发明公开了一株深绿木霉（Trichoderma atroviride）B-8-1-34在制备漆酶方面的应用，属于微生物技术领域。该菌株已于2012年8月10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，保藏编号为：CGMCC No. 6390。利用该菌株通过液态发酵法生产漆酶，通过调整培养基成份提高漆酶产量。液态发酵周期短，酶活高，酶稳定性好，为漆酶的生产及在食品、环保等领域的应用提供了新的菌种资源。
Description
技术领域
本发明涉及一株深绿木霉B-8-1-34菌株制备漆酶方面的应用，属于微生物技术领域。
背景技术
真菌漆酶是一种糖蛋白，由肽链、糖配基和Cu2+三个部分组成。分子量在60~390kDa 之间。肽链一般由500~550个氨基酸组成，糖配基主要有氨基己糖、葡萄糖、甘露糖、半乳糖、岩藻糖和阿拉伯糖，占整个分子重量的10%~80%。糖配基组成及含量的不同是漆酶分子量存在较大差异的主要原因。由于漆酶具有底物多样性，因此，具有广泛的工业应用价值和潜力。如添加到酒类制品、果汁、饮料及食用油中可抗氧化；添加到洗涤剂中可提高洗涤效果，还可用于纸浆生物漂白，废水处理，环境污染物脱毒与降解，以及染发剂。此外，还可用于聚合和氧化反应的催化剂或促进剂等。
漆酶可由多种真菌产生，包括子囊菌类（如曲霉属、链孢霉属和柄孢壳菌属）、半知菌类葡萄孢属、担子菌类（如金线菌属、屈孔菌属、 香菇属、灰侧耳菌属、栓菌属）和丝核菌属。木霉属菌株可产生纤维素酶、几丁质酶和木聚糖酶等多种具有生物活性或重要工业应用价值的酶。然而，利用木霉属菌株发酵产漆酶鲜有报道。法国学者Savoie研究了木霉对7株叶腐担子菌和7株木腐担子菌漆酶合成能力的影响，木霉使酶活均有所提高。Baldrian 以木霉与云芝共培养产生的酶活比对照提高40倍，达44.2U/L; Mata 报道以咖啡果肉为基质共培养平菇和木霉，酶活从150U/g 提高到180 U/g。CN200610037937.1 公开了利用栓菌AH28-2和木霉ZH1共培养生产漆酶的方法，发酵9.5天，漆酶产量达6000U/L。Hanen Chakroun等人以葡萄糖为碳源并加入适量铜离子培养深绿木霉，漆酶产量可达到661U/L; U. Holker研究了深绿木霉产漆酶的影响条件，其中，当pH=4.5时酶活最高，为7U/L。
发明内容
本发明的目的是利用保藏号为CGMCC No. 6390的深绿木霉 菌株高产量地制备稳定性好的漆酶。
为实现本发明目的，本发明利用深绿木霉菌株通过液态发酵法生产漆酶，通过调整培养基成份提高漆酶产量。该菌株是以木糖渣为唯一碳源，从自然界中筛选出，经分子鉴定，结合形态学特征确定该菌株为深绿木霉，进一步经过诱变选育获得。已于2012年8月10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（北京市朝阳区北辰西路1号院3号），保藏号为CGMCC No. 6390。
具体制备方法如下：
⑴将保藏号为CGMCC No. 6390的深绿木霉B-8-1-34经活化后，制成浓度为106～108/ml的孢子悬液，按1～10%的接种量接入种子培养基中，26～32℃，150～220rpm摇床中培养24-48h，得种子液；种子培养基：PDA液体培养基，121℃高压灭菌25 min；
⑵将步骤⑴制得的种子液以2～15%接种量接入发酵产酶培养基中，裝液量为30～60mL/250mL, 26～32℃，150～220rmp摇床中培养；
⑶培养48～120h，固液分离制备粗酶液，或将制得的粗酶液浓缩后，经过常规提取得到固体酶制剂。
步骤（2）所述的发酵产酶培养基由以下组份组成：碳源0.5～5%，氮源0.5～5%，磷酸二氢钾0.01～0.4%；七水硫酸镁0.002～0.10%；氯化钾0.002～0.10%，各组分以重量百分比计，余量为蒸馏水；并加入CuSO4 和微量元素，使培养基中CuSO4 50～500μmol/L，微量元素液1～5mL/L。
所述的碳源为甘油、葡萄糖、蔗糖、麦芽糖、木糖、果糖、乳糖、山梨糖、甘露糖、可湿淀粉、玉米秸秆其中之一或两种以上成份的混合。
所述的氮源为酵母提取物、蛋白胨、麸皮、豆饼粉、玉米浆、碳酸氢铵、酒石酸铵、磷酸氢二铵、硫酸铵、氯化铵、酒石酸铵、硝酸铵其中之一或两种以上成份的混合。
所述的碳源优选果糖、甘露糖和蔗糖、可湿淀粉其中之一或两种以上成份的混合。
所述的氮源优选蛋白胨、酵母提取物、硝酸铵、磷酸氢二铵其中之一或两种以上成份的混合。
所述的微量元素液组成为每L含有如下成份：B4O7Na2 ·10H2 O 0.1；CuSO4 ·5H2O 0.01；FeSO4 ·7H2O 0.05；MnSO4 ·7H2O 0.01；ZnSO4 ·7H2O 0.07；(NH4)6MO7O24 ·4H2O 0.01，以g计。
步骤（2）在发酵过程中可以适量添加表面活性剂，以提高漆酶的产量。添加方式可以于接种前或发酵24h后；添加量为底物重量的0.0001～0.001%。
所述的表面活性剂主要有:Tween-20、Tween-40、Tween-60、Tween-80、
TritonX-100、TritonX-114、TritonX-116、TritonX-405、TX-4、TX-6、TX-9、TX-10、AEO-3、AEO-7、AEO-9、一乙醇胺、二乙醇胺、三乙醇胺等。
所述的深绿木霉发酵生产漆酶的方法，添加Zn2+、Co2+、Fe3+、Ca2+、K+、 Li+可以提高深绿木霉的漆酶产量。
酶活测定：以2,2 -联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)为底物，反应在室温下进行，在总反应体积3mL中，含有0.5mL ABTS（5mmol/L），2mL 100 mmol/L柠檬酸酸-柠檬酸钠缓冲液（pH5.0），添加0.5mL的酶液（或适当稀释的酶粉）启动反应，在420nm下反应5min，测定吸光值的变化，以柠檬酸-柠檬酸钠缓冲液为对照。
酶活定义为：每分钟转化1μmol底物或形成1μmol产物所需要的酶量为1个酶活单位(U)，ε420＝3.6×104[(mol/L)-1cm-1]。
本发明利用深绿木霉菌株CGMCC No. 6390，通过液态发酵法生产漆酶，其液体产酶发酵最高酶活达1700U/L。该菌株液态发酵具有产酶能力强，发酵周期短，所产漆酶稳定性好等特点，非常适合工业化应用。
附图说明
图1为本发明实施例制备的粗酶液经稀释后，采用不同pH 的缓冲液，室温测定，酶活变化曲线；
图2为本发明实施例制备的粗酶液经稀释后，不同温度测定，酶活变化曲线；
图3为本发明实施例制备的粗酶液经稀释后，在不同pH 的缓冲液中室温处理6h测定，剩余酶活变化曲线；
图4为为本发明实施例制备的粗酶液经稀释后，在不同温度下处理6h测定，剩余酶活变化曲线。
具体实施方式
为对本发明进行更好的说明，举实施例如下：
实施例1深绿木霉菌株CGMCC No. 6390，发酵产漆酶可利用的碳源
按照上述方法，步骤（2）培养基组成为（g/L）: 碳源20；酵母提取物5；酒石酸铵5；磷酸二氢钾1；七水硫酸镁0.5；氯化钾0.5；CuSO4 300μmol/L；微量元素液3ml/L; 微量元素液（g/L）：B4O7Na2 ·10H2O 0.1；CuSO4 ·5H2O 0.01；FeSO4 ·7H2O 0.05；MnSO4 ·7H2O 0.01；ZnSO4 ·7H2O 0.07；(NH4)6MO7O24·4H2O 0.01。pH自然，摇瓶装量为30mL/250mL,接种量为10%，种龄28h，30℃，180rpm发酵，定期取样测定。碳源分别为甘油、葡萄糖、蔗糖、麦芽糖、木糖、果糖、乳糖、山梨糖、甘露糖、可湿淀粉、玉米秸秆。漆酶产量如表1所示。
表1 碳源对漆酶产量的影响
| 碳源 | 发酵时间（h） | 酶活（U/L） | 碳源 | 发酵时间（h） | 酶活（U/L） |
| 甘油 | 96 | 200 | 乳糖 | 72 | 160 |
| 葡萄糖 | 70 | 24 | 山梨糖 | 72 | 700 |
| 蔗糖 | 70 | 293 | 甘露糖 | 72 | 1260 |
| 麦芽糖 | 72 | 400 | 可湿淀粉 | 72 | 1480 |
| 木糖 | 120 | 640 | 果糖 | 120 | 1700 |
| 玉米秸秆 | 72 | 360 |
实施例2氮源对B8-1-34发酵产漆酶的影响
培养基组成为（g/L）: 有机氮5；蔗糖20；无机氮5；磷酸二氢钾1；七水硫酸镁0.5；氯化钾0.5；微量元素液3mL/L; CuSO4 300μmol/L；pH自然。摇瓶装量为30mL/250mL,接种量为10%，种龄24小时， 30℃，180rpm 发酵。所用的有机氮、无机氮见表2，微量元素液组成同实施例1。漆酶产量如表2所示。
表2氮源对B8-1-34发酵产漆酶的影响
| 氮源1 | 发酵时间（h） | 酶活（U/L） | 氮源2 | 发酵时间（h） | 酶活（U/L） |
| 酵母提取物 | 48 | 60 | 碳酸氢铵 | 96 | 146 |
| 蛋白胨 | 72 | 1332 | 酒石酸铵 | 96 | 93 |
| 麸皮 | 72 | 7 | 磷酸氢二铵 | 72 | 266 |
| 豆饼粉 | 96 | 224 | 硫酸铵 | 72 | 306 |
| 玉米浆 | 72 | 226 | 氯化铵 | 72 | 706 |
| 硝酸铵 | 72 | 626 |
注：氮源1处理另加0.5%酒石酸铵；氮源2处理另加0.5%酵母提取物
实施例3 最佳pH、温度及pH和温度耐受情况
如上制备的粗酶液经稀释后，采用不同pH 的缓冲液，室温测定酶活, 以酶活最高的测定值为参照（100%），计算相对酶活，结果如图1所示；如上制备的粗酶液经稀释后，于不同温度下测定酶活，以酶活最高的测定值为参照（100%），计算相对酶活。结果如图2所示。
如上制备的粗酶液经稀释后，在不同pH 的缓冲液中室温处理6h，分析测定剩余酶活，结果如图3所示；如上制备的粗酶液经稀释后，于不同温度处理6h，分析测定剩余酶活，结果如图4所示。
实施例4
⑴将保藏号为CGMCC No. 6390的深绿木霉B-8-1-34经活化后，制成浓度为106～108/ml的孢子悬液，按10%的接种量接入种子培养基中， 30℃，150～220rpm摇床中培养40h，得种子液；种子培养基：PDA液体培养基，121℃高压灭菌25 min；
⑵将步骤⑴制得的种子液以5%接种量接入发酵产酶培养基中，裝液量为40mL/250mL, 28℃，150～220rmp摇床中培养；发酵24h后添加表面活性剂Tween-20或Tween-40，添加量为底物重量的0.001%。
⑶步骤（2）所述的发酵产酶培养基由以下组份组成：碳源1.5%，氮源1%，磷酸二氢钾0.2%；七水硫酸镁0.05%；氯化钾0.1%，各组分以重量百分比计，余量为蒸馏水；并加入CuSO4 和微量元素，使培养基中CuSO4 200μmol/L，微量元素液3mL/L。
所述的碳源为果糖。
所述的氮源为蛋白胨和酒石酸铵以10：1重量份的混合物。
所述的微量元素液组成为每L含有如下成份：B4O7Na2 ·10H2 O 0.1；CuSO4 ·5H2O 0.01；FeSO4 ·7H2O 0.05；MnSO4 ·7H2O 0.01；ZnSO4 ·7H2O 0.07；(NH4)6MO7O24 ·4H2O 0.01，以g计。
培养72h，固液分离制备粗酶液，测得漆酶酶活为 739 U/L 。
实施例5
步骤（2）发酵过程中添加Zn2+、Co2+、Fe3+、Ca2+、K+ 和Li+，加量为0.005mmol/L；所述的氮源选蛋白胨和酵母提取物两种以上成份的混合。其它如实施例4，测得漆酶酶活为 857 U/L 。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.深绿木霉菌株B-8-1-34在制备漆酶方面的应用，其特征在于：
(1)将保藏号为CGMCC No. 6390的深绿木霉（Trichoderma atroviride）B-8-1-34经活化后，制成浓度为106～108/mL的孢子悬液，按1～10%的接种量接入种子培养基中，26～32℃，150～220rpm摇床中培养24～48h，得种子液；种子培养基：PDA液体培养基，121℃高压灭菌25 min；
(2)将步骤(1)制得的种子液以2～15%接种量接入发酵产酶培养基中，裝液量为30～60mL/250mL, 26～32℃，150～220rmp摇床中培养；
(3)培养48～120h，固液分离制备粗酶液，或将制得的粗酶液浓缩后，经过常规提取得到固体酶制剂；
步骤（2）所述的发酵产酶培养基由以下组份组成：碳源0.5～5%，氮源0.5～5%，磷酸二氢钾0.01～0.4%；七水硫酸镁0.002～0.10%；氯化钾0.002～0.10%，各组分以重量百分比计，余量为蒸馏水；并加入CuSO4 和微量元素，使培养基中CuSO4 50～500μmol/L，微量元素液1～5mL/L；
所述的碳源为甘油、葡萄糖、蔗糖、麦芽糖、木糖、果糖、乳糖、山梨糖、甘露糖、可湿淀粉、玉米秸秆其中之一或两种以上成份的混合；
所述的氮源由有机氮和无机氮组成，氮源选自酵母提取物、蛋白胨、麸皮、豆饼粉、玉米浆酒石酸铵、碳酸氢铵、酒石酸铵、磷酸氢二铵、硫酸铵、氯化铵或硝酸铵；
所述的微量元素液组成为每L含有如下成份：B4O7Na2 ·10H2 O 0.1；CuSO4 ·5H2O 0.01；FeSO4 ·7H2O 0.05；MnSO4 ·7H2O 0.01；ZnSO4 ·7H2O 0.07；(NH4)6MO7O24 ·4H2O 0.01，以g计。
2.如权利要求1所述的深绿木霉菌株B-8-1-34在制备漆酶方面的应用，其特征在于：所述的碳源优选果糖、甘露糖和蔗糖、可湿淀粉其中之一或两种以上成份的混合；
所述的氮源优选蛋白胨、酵母提取物其中之一与酒石酸铵混合物或硝酸铵、酒石酸铵、磷酸氢二铵其中之一与酵母提取物混合物。
3.如权利要求1或2所述的深绿木霉菌株B-8-1-34在制备漆酶方面的应用，其特征在于：步骤（2）发酵过程中于接种前或发酵24h后，添加表面活性剂，添加量为底物重量的0.0001～0.001%。
4.如权利要求1或2所述的深绿木霉菌株B-8-1-34在制备漆酶方面的应用，其特征在于：步骤（2）发酵过程中添加Zn2+、Co2+、Fe3+、Ca2+、K+或/和Li+。
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| Extracellular Laccases in Ascomycetes Trichoderma atroviride and Trichoderma harzianum;U. HOLKER, J. DOHSE, M. HOFER;《Folia Microbiol》;20021231;第47卷(第4期);全文 * |
| Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds;H. Chakroun et al.;《Process Biochemistry》;20101231;全文 * |
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