CN114410536B - 一种细菌培养释放胞内酶的方法 - Google Patents
一种细菌培养释放胞内酶的方法 Download PDFInfo
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Abstract
本发明涉及一种细菌培养释放胞内酶的方法,所述方法包括以下步骤:在无菌条件下,从芽孢杆菌菌种保存斜面中挑取一环,接入液体培养基中,30‑37℃下转速180r/min振荡培养24‑48h,当芽孢杆菌培养到一定的OD值时,对其进行胁迫处理,使菌体破裂,从而释放出胞内酶。本方法有效解决胞内酶释放过程中破坏酶活性、成本高、溶剂残留等种种技术难题,实现了胞内酶的快速高效释放。富含胞内酶的细菌培养液可高效降解木质素、促进氮和碳的转化,将有机物及难降解物质如木质素纤维素原料快速降解,可用于发酵降解农业废弃物;也可与发酵菌和胞外酶协同作用于秸秆、尾菜、果蔬园林枝条、厨余垃圾等发酵堆肥及秸秆饲料化。
Description
技术领域
本申请涉及农业废弃物发酵剂制备领域,具体涉及一种细菌培养释放胞内酶的方法。
背景技术
农业废弃物常被发酵处理后资源再利用,但是常用农业废弃物发酵技术不能找到有效分解高木质素和高氮原料,原料发酵时间长,未能精准控制腐植酸大分子的合成,不能有效解决微生物分解过程中出现的碳氮气体高排放量、堆肥得率低、堆肥中腐植酸含量少的问题。
目前,采用酶菌复合制剂进行农业废弃物发酵获得再生资源,是本领域的常用技术手段,但是各种酶制剂的制备方法复杂,经济成本过高,酶菌复合制剂的配置效果并不理想。
胞内酶是在细胞内具有一定催化作用的酶,这些酶在细胞内常与颗粒体结合并有着一定的分布。例如过氧化物酶,广泛分布于植物的各种组织中,并参与其生理活动,是植物体内具有防御作用的成员。过氧化物酶也是一种重要的植物保护酶,可清除逆境产生的毒性活性氧。
目前常用酶制剂绝大多数为胞外游离酶,而胞内酶具有胞外酶所不具备的特点,具有强大的分解能力,但一般只在胞内产生。另外,细菌的有些酶也只有在细胞分解时才释放。胞内酶的开发对自然界有机物的分解具有重要意义。
利用微生物来生产酶在酶制剂的生产方法中占有重要的地位,特别是绿色高效的胞内酶释放方法是酶制剂的生产过程中至关重要。目前细胞破碎的方法主要可分为机械技术(如高压均质、珠磨)以及非机械技术(物理法、化学法及酶法)。另外,通过将裂解基因导入细胞,可以实现细胞的诱导裂解。物理法主要依靠机械作用,使细胞遭到强大的机械剪切力而被破坏,但机械剪切力会破坏酶的结构及活性。酶法处理是一种在实验室取得广泛应用的有效手段,但溶菌酶的高成本限制了其工业化应用。化学法主要使用有机试剂或化学渗透剂,如表面活性剂等。人工合成表面活性剂在破碎液中难去除,造成下游纯化处理困难。
因此,研究一种通过细菌培养快速高效释放胞内酶的方法,获得含有胞内酶的细菌培养液,解决胞内酶释放过程酶活性被破坏、成本高、溶剂残留等种种技术难题,最终通过胞内酶的作用,有效将农林废弃物循环利用,对促进农业可持续发展均具有重要意义。
发明内容
本申请的发明目的是:通过常规培养细菌,达到一定的OD值后对细菌进行胁迫培养,使得细菌菌体破裂释放出胞内酶,实现了胞内酶的快速高效释放,含有胞内酶的细菌培养液可以促进发酵底物氮和碳的转化,将木质素原料和高氮原料快速降解,特别适用于发酵降解农业废弃物,如园林修剪枝,强化发酵过程中木质素类物质转化成腐植酸前体-醌类物质的比例,大大提高了堆肥中腐植酸的含量。解决胞内酶释放过程破坏酶活性、成本高、溶剂残留等技术问题。
为实现上述发明目的,解决上述技术问题,本发明提供一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30-37℃下转速180r/min振荡培养24-48h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,胞内酶释放出来;
进一步的,所述芽孢杆菌为枯草芽孢杆菌、地衣芽孢杆菌中的一种;
进一步的,所述步骤(1)中的培养温度为30℃或35℃或37℃;
进一步的,所述步骤(1)中的培养时间为24h或36h或48h;
进一步的,所述步骤(2)中的OD值为6-8;
更进一步的,胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入占液体培养基4.5-5.5%的氯化铵或硫酸铵,继续培养5-8h。
进一步的,所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min。一种细菌培养释放胞内酶的方法制备而成的含胞内酶的细菌培养液。
进一步的,获得一种细菌培养释放胞内酶的方法制备而成的含胞内酶的细菌培养液。
更进一步的,将细菌培养释放胞内酶的方法制备而成的含胞内酶的细菌培养液用于发酵降解农业废弃物,所述农业废弃物优选园林修剪枝。
本发明的有益效果:
本发明能够促进胞内酶的快速高效释放,促进氮和碳的转化,将木质素原料快速降解,可用于发酵降解农业废弃物,如园林修剪枝,芽孢杆菌胞内酶也为菌的生长进行有效调控,同时实现高木质素原料快速降解,强化发酵过程中木质素类物质转化成腐植酸前体-醌类物质的比例,大大提高了堆肥中腐植酸的含量。本发明相比于传统的物理法、化学法及酶法细胞破碎,提高了酶活性,降低了成本,无溶剂残留,适用于工业化生产,促进了农业可持续发展。芽孢杆菌胞内酶促使难降解的高木质素原料降解效率更高;也可与发酵菌和胞外酶协同作用于秸秆、尾菜、果蔬园林枝条、厨余垃圾等发酵堆肥及秸秆饲料化。
附图说明
图1芽孢杆菌胞内酶粗提液过氧化物酶酶活测定实验结果。
图2芽孢杆菌胞内酶粗提液肽聚糖酶酶活测定实验结果。
具体实施方式
实施例1
一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30℃下转速180r/min振荡培养24h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,胞内酶释放出来;
进一步的,所述芽孢杆菌为枯草芽孢杆菌;
进一步的,所述步骤(2)中的OD值为6;
更进一步的,胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入5%氯化铵,继续培养5h。
进一步的,所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min。
实施例2
一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30℃下转速180r/min振荡培养24h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,胞内酶释放出来;
进一步的,所述芽孢杆菌为地衣芽孢杆菌;
进一步的,所述步骤(2)中的OD值为6;
更进一步的,胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入5%硫酸铵,继续培养5h。
进一步的,所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min。
实施例3
一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30℃下转速180r/min振荡培养24h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,胞内酶释放出来;
进一步的,所述芽孢杆菌为枯草芽孢杆菌;
进一步的,所述步骤(2)中的OD值为8;
更进一步的,胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入5%氯化铵,继续培养5h。
进一步的,所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min。
实施例4
一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30℃下转速180r/min振荡培养24h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,胞内酶释放出来;
进一步的,所述芽孢杆菌为枯草芽孢杆菌;
进一步的,所述步骤(2)中的OD值为5;
更进一步的,胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入5%硫酸铵,继续培养5h。
进一步的,所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min。
实施例5
一种细菌培养释放胞内酶的放大规模化生产方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入500ml液体培养基中,30℃下转速180r/min振荡培养24h;
(2)当芽孢杆菌(1)培养到一定的OD值时,接种至50L发酵槽的液体培养基中30℃下转速150~250rpm通气量0.5~1.5vvm培养6~10h;
(3)当芽孢杆菌(2)培养到一定的OD值时,接种至500L发酵槽的液体培养基中30℃下转速75~100rpm通气量0.5~1.5vvm培养6~10h;
(4)当芽孢杆菌(3)培养到一定的OD值时,接种至5000L发酵槽的液体培养基中30℃下转速30~50rpm通气量0.5~1.5vvm背压0.2kg/cm2培养10~18h;
(5)当芽孢杆菌(4)培养到一定的OD值时,停止供气并将温度提升至50℃下培养20min,加入5%硫酸铵或氯化铵,继续培养8h;
(6)当芽孢杆菌(5)胁迫培养加入适量的硅藻土后使用板框过滤机进行固液分离;
(7)固液分离完的含胞内酶的滤液(6)加入适量的麦芽糊精进行喷雾干燥;
进一步的,所述芽孢杆菌为枯草芽孢杆菌;
进一步的,所述步骤(2)中的OD值为5~8;
进一步的,所述步骤(3)中的OD值为5~8;
进一步的,所述步骤(4)中的OD值为5~8;
进一步的,所述步骤(5)中的OD值为10;
进一步的,所述步骤(1)中的液体培养基为:蛋白胨5g,酵母提取物2.5g,氯化钠5g,蒸馏水500mL,pH 7.0-7.2,121℃灭菌20min。
进一步的,所述步骤(2)中的液体培养基为:蛋白胨0.5kg,酵母提取物0.5kg,氯化钠0.5kg,磷酸氢二钾0.5kg磷酸二氢钾0.5kg硫酸铵1kg硫酸镁0.5kg消泡剂0.05kg自来水50L,pH 7.0-7.2,121℃灭菌20min。
进一步的,所述步骤(3)中的液体培养基为:酵母提取物5kg,氯化钠5kg,磷酸氢二钾5kg磷酸二氢钾5kg硫酸铵10kg硫酸镁5kg消泡剂0.5kg自来水500L,pH 7.0-7.2,121℃灭菌20min。
进一步的,所述步骤(4)中的液体培养基为:酵母提取物50kg,氯化钠50kg,磷酸氢二钾50kg磷酸二氢钾50kg硫酸铵100kg硫酸镁50kg消泡剂5kg自来水5000L,pH 7.0-7.2,121℃灭菌20min。
进一步的,所述步骤(5)中的升温速度为每分钟0.5-1℃。
进一步的,所述步骤(6)中的硅藻土量为50kg。板框滤膜孔隙为1微米。
进一步的,所述步骤(7)中的麦芽糊精量为1000kg为:喷雾干燥温温度入风为350~400℃,最终成品水分<5%。
实施例五实现了胞内酶产品的工业化生产,增加产品稳定性,降低成本,实现量产,以满足实际生产的需要。本申请的申请人发现随着发酵槽体积加大,通过调控氧气,把大气泡变成小气泡,控制通氧量,使菌体边破裂边合成,达成动态,从而解决了量产的技术难题。
实验一:芽孢杆菌胞内酶粗提液过氧化物酶酶活测定实验
实验例1-4:将实施例1-4所得菌液7000r/min离心15min,收集上清液,过滤,得到胞内酶粗酶液。
空白例:省略步骤(2)的胁迫培养,其他同实施例1。
对比例1:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%的EDTA和蔗糖混合溶液,继续培养5h,再按照实验例1的方法制得粗酶液。混合溶液中EDTA溶液的浓度为0.05mol/L,蔗糖溶液浓度为0.5mol/L,二者体积比为1:1。
对比例2:将实施例1的胁迫培养方法替换为仅停止供氧,隔绝氧气,在50℃下培养20min,再按照实验例1的方法制得粗酶液。
对比例3:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%氯化铵,继续培养5h,再按照实验例1的方法制得粗酶液。
对比例4:将实施例1的胁迫培养方法替换为将培养好的菌液加入溶菌酶达到终浓度0.5g/L,继续培养5h,再按照实验例1的方法制得粗酶液。其中溶菌酶溶液采用pH=8,终浓度分别为20mM和2mM的Tris和EDTA-Na2缓冲液配制。
试验方法:
采用愈创木酚法测定酶活,取2.95mL 0.15mol/L pH6.0的磷酸缓冲液,加入0.03mL 30%过氧化氢,0.02mL愈创木酚溶液于比色皿中,再加入0.3mL粗酶液,于30℃水浴条件下预热20s后,于470nm下测定吸光度,每分钟吸光值变化0.001为一个酶活力单位(U),以实施例1的酶活为标准,取同样的温度和时间点测定每组的相对酶活。
实验结果:
实施例1-4相比于空白例酶活显著提高,证明了本发明的胞内酶培养方法能够让细胞内的过氧化物酶等胞内酶高效释放。本申请的胁迫方法相比于传统的物理渗透压冲击法和酶溶液破碎法(对比例1、4)能够更好地促进胞内酶的释放。在OD值达到6-8时,对枯草芽孢杆菌胁迫效果高于OD值为5时。
实验二:芽孢杆菌胞内酶粗提液肽聚糖酶酶活测定实验
实验例1-4:将实施例1-4所得菌液7000r/min离心15min,收集上清液,过滤,得到胞内酶粗酶液。
空白对比例:省略步骤(2)的胁迫培养,其他同实施例1。
对比例1:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%的EDTA和蔗糖混合溶液,继续培养5h,再按照实验例1的方法制得粗酶液。混合溶液中EDTA溶液的浓度为0.05mol/L,蔗糖溶液浓度为0.5mol/L,二者体积比为1:1。
对比例2:将实施例1的胁迫培养方法替换为仅停止供氧,隔绝氧气,在50℃下培养20min,再按照实验例1的方法制得粗酶液。
对比例3:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%氯化铵,继续培养5h,再按照实验例1的方法制得粗酶液。
对比例4:将实施例1的胁迫培养方法替换为将培养好的菌液加入溶菌酶达到终浓度0.5g/L,继续培养5h,再按照实验例1的方法制得粗酶液。其中溶菌酶溶液采用pH=8,终浓度分别为20mM和2mM的Tris和EDTA-Na2缓冲液配制。
试验方法:
1)制备金黄色葡萄球菌菌悬液:接种金黄色葡萄球菌到LB液体培养基中,25℃、200rpm/min条件下震荡培养12h。吸取1ml培养物离心收集菌体,再用1ml 0.5mM的PBS缓冲液重悬制备葡萄球菌菌悬液(浓度大约107CFU/ml)。
2)吸取200μl制备好的菌悬液,与200μl粗酶液混匀,30℃反应40min;空白组加入经沸水浴1min失活的粗酶液,其他与实验组相同。
3)反应后,将空白组与实验组反应液用LB培养基进行梯度稀释到1×10-5、1×10-6、1×10-7,然后分别取0.1ml涂布固体LB平板,37℃恒温倒置培养24hr计数空白组与实验组的菌落个数。
以抑菌率(葡萄球菌被降解)来表示酶活:
抑菌率=(菌落数(空白)-菌落数(处理))/(菌落数(对照))
实验结果:
实施例1-4相比于空白例抑菌率显著提高,说明实施例1-4肽聚糖酶的酶活显著提高,肽聚糖酶属于胞内酶,证明了本发明的胞内酶培养方法能够让细胞内的肽聚糖酶高效释放。本申请的胁迫方法相比于传统的物理渗透压冲击法和酶溶液破碎法(对比例1、4)能够更好地促进胞内酶的释放。在OD值达到6-8时,对枯草芽孢杆菌胁迫效果高于OD值为5时。
实验三:芽孢杆菌的木质素降解实验
实验例1-4:实施例1-4所得菌液。
空白例:省略步骤(2)的胁迫培养,其他同实施例1。
对比例1:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%的EDTA和蔗糖混合溶液,继续培养5h。混合溶液中EDTA溶液的浓度为0.05mol/L,蔗糖溶液浓度为0.5mol/L,二者体积比为1:1。
对比例2:将实施例1的胁迫培养方法替换为仅停止供氧,隔绝氧气,在50℃下培养20min。
对比例3:将实施例1的胁迫培养方法替换为将培养好的菌液加入5%氯化铵,继续培养5h。
对比例4:将实施例1的胁迫培养方法替换为将培养好的菌液加入溶菌酶达到终浓度0.5g/L,继续培养5h。其中溶菌酶溶液采用pH=8,终浓度分别为20mM和2mM的Tris和EDTA-Na2缓冲液配制。
试验方法:
将实验例、对照例进行碱木质素液体培养,以对碱木质素溶液的降解率为标准量化粗酶液对木质素降解能力:分别按照10%的接种比将酶菌复合发酵剂接种到碱木质素降解培养基中,30℃、150r/min培养10d,测定每天碱木质素降解率。碱木质素降解培养基:碱木质素1.0g,NH3Cl 2.0g,K2HPO41.0 g,KH2PO41.0 g,MgSO4·7H2O 0.2g,CaCl20.1 g,FeSO4·7H2O 0.05g,MnSO4·7H2O 0.02g,琼脂15.0g,补水至1000mL,121℃灭菌20min。
实验结果:
表1木质素降解结果
降解率(%) | 1d | 5d | 10d |
实验例1 | 45.1 | 48.9 | 61.6 |
实验例2 | 43.7 | 46.5 | 58 |
实验例3 | 47.3 | 50.2 | 64.5 |
实验例4 | 42.4 | 45 | 58.8 |
空白例 | 23.5 | 27.8 | 35.2 |
对比例1 | 30.1 | 33.5 | 46.4 |
对比例2 | 38.3 | 44.6 | 55.6 |
对比例3 | 37.1 | 42.2 | 54.7 |
对比例4 | 33.7 | 38.9 | 51.2 |
结果显示,实验例1-4降解碱木质素速度更快,碱木质素10d降解率更高。由此说明,实施例1-4的方法培养后的芽孢杆菌降解碱木质素速度更快,最高降解率更高。该实验表明实施例培养方法释放的胞内酶对园林修剪枝的碱木质素降解效率很高。
实验四:氯化铵用量选择实验
实验例1:将实施例1所得菌液7000r/min离心15min,收集上清液,过滤,得到胞内酶粗酶液。
对比例1:将实施例1的胁迫培养方法中氯化铵的添加量改为3%,其他条件不变。
对比例2:将实施例1的胁迫培养方法中氯化铵的添加量改为7%,其他条件不变。
对比例3:将实施例1的胁迫培养方法中氯化铵的添加量改为9%,其他条件不变。
试验方法:参照实验一和实验二的方法。
实验结果:
表2氯化铵用量选择实验结果
相对酶活(%) | 抑菌率(%) | |
实验例1 | 100 | 88±1 |
对比例1 | 92±2 | 80±3 |
对比例2 | 97±1 | 85±2 |
对比例3 | 95±2 | 86±1 |
结果显示,氯化铵用量在5%能够达到相对酶活的最大值,用量过多或过少均会影响相对酶活,而抑菌率用量5%以上差别不大,从成本考虑选择5%作为最优添加量。
实验五:硫酸铵用量选择实验
实验例2:将实施例2所得菌液7000r/min离心15min,收集上清液,过滤,得到胞内酶粗酶液。
对比例1:将实施例2的胁迫培养方法中硫酸铵的添加量改为3%,其他条件不变。
对比例2:将实施例2的胁迫培养方法中硫酸铵的添加量改为7%,其他条件不变。
对比例3:将实施例2的胁迫培养方法中硫酸铵的添加量改为9%,其他条件不变。
试验方法:参照实验一和实验二的方法。
实验结果:
表3硫酸铵用量选择实验结果
相对酶活(%) | 抑菌率(%) | |
实验例2 | 105±2 | 83±2 |
对比例1 | 95±3 | 74±3 |
对比例2 | 102±1 | 80±1 |
对比例3 | 100±2 | 78±3 |
结果显示,硫酸铵用量在5%能够达到相对酶活的最大值,用量过多或过少均会影响相对酶活,而抑菌率用量5%以上差别不大,从成本考虑选择5%作为最优添加量。
以上描述了本发明优选实施方式,然其并非用以限定本发明。本领域技术人员对在此公开的实施方案可进行并不偏离本发明范畴和精神的改进和变化。
Claims (3)
1.一种细菌培养释放胞内酶的方法,其特征在于:所述方法包括以下步骤:
(1)在无菌条件下,从芽孢杆菌菌种保存斜面中挑取菌种,接入液体培养基中,30-37℃下转速180r/min振荡培养24-48h;
(2)当芽孢杆菌培养到一定的OD值时,对其进行胁迫培养,使菌体破裂,从而释放出胞内酶;
所述芽孢杆菌为枯草芽孢杆菌、地衣芽孢杆菌中的一种;
所述步骤(2)中的OD值为6-8;
所述步骤(1)中的液体培养基为:蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水1000mL,pH 7.0-7.2,121℃灭菌20min;
所述胁迫培养方法如下:停止供氧,隔绝氧气,在50℃下培养20min,加入占液体培养基4.5-5.5%的氯化铵或硫酸铵,继续培养5-8h。
2.如权利要求1所述的一种细菌培养释放胞内酶的方法,其特征在于,所述步骤(1)中的培养温度为30℃或35℃或37℃。
3.如权利要求1所述的一种细菌培养释放胞内酶的方法,其特征在于,所述步骤(1)中的培养时间为24h或36h或48h。
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