CN103131681A - 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B8-1-34(TrichodermaatrovirideB8-1-34)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:CGMCCNo.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B8-1-34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B8-1-34(TrichodermaatrovirideB8-1-34)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B8-1-34(TrichodermaatrovirideB8-1-34)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(Trichodermaatroviride),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B8-1-34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B8-1-34(TrichodermaatrovirideB8-1-34)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B8-1-34(TrichodermaatrovirideB8-1-34)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B8-1-34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 (5)
1.深绿木霉菌株B8-1-34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B8-1-34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;
(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、硝酸铵其中之一或两种以上成份的混合。
2.如权利要求1所述的深绿木霉菌株B8-1-34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、硝酸铵、酒石酸铵、磷酸氢二铵其中之一或两种以上成份的混合。
3.如权利要求1或2所述的深绿木霉菌株B8-1-34在制备漆酶方面的应用,其特征在于:所述的微量元素液组成为每L含有如下成份: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,以g计。
4.如权利要求1或2所述的深绿木霉菌株B8-1-34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%。
5.如权利要求1或2所述的深绿木霉菌株B8-1-34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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