用海洋细菌Pseudoalteromonas tetrodonis QZ-4发酵生产褐藻胶裂解酶的研究
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摘要
褐藻胶裂解酶在褐藻胶的生物降解、海藻原生质体制备以及治疗纤维化囊肿等方面有着重要的用途,特别在降解褐藻胶制备多种活性产物方面有着广阔的前景和实际生产意义。本研究分离到了一株新型产褐藻胶裂解酶的海洋细菌P. terodonis. QZ-4;在对其产酶培养基和培养条件进行优化的基础上,建立了分批发酵动力学模型；并探讨了以褐藻胶为底物发酵所得的褐藻胶裂解酶的纯化、酶学性质和酶促反应动力学；建立用粗酶液降解褐藻胶的方法；此外,还初步探讨了褐藻胶酶解糖液对玉米等萌发的影响。主要研究结论如下：
1.在浙江舟山、山东青岛、西班牙等沿海地区采集样品,共采集海水、海藻、泥沙等样品58份,通过平板粗筛得到大量细菌、放线菌、酵母和霉菌,其中具有酶活性的细菌计483株、霉菌计1株；对这484株菌进行平板扩散筛选,结合摇瓶初筛和复筛手段,得到一株生长和产酶(70U/mL)较稳定的海洋细菌。该菌在2.5%NaCl浓度的分离筛选平板上,28℃培养1d,菌落为白色,圆形(Φ1mm),表面湿润光滑,不产色素,革兰氏染色阴性,菌体形态呈短杆状(0.2-0.4μmm×0.7-1μm),单端带鞭毛。通过16S rDNA鉴定,序列G+C含量为52.2%,BLAST程序比对,该菌株与Pseudoalteromonas tetrodonis相似性为97%,命名为Pseudoalteromonas terodonis QZ-4。
27采用部分因子实验方法和响应面法对P. terodonis QZ-4发酵生产褐藻胶裂解酶的培养基条件进行优化。首先明确了磷酸氢二铵、氯化钠和硫酸亚铁是影响褐藻胶裂解酶活性的主要因素,进而用最陡爬坡路径逼近最大响应区域,并采用中心组合设计及响应面分析确定了主要影响因子的最佳浓度,最终获得的优化培养基组成为(g/L)：褐藻酸钠5、磷酸氢二铵11.6、氯化钠32.9、硫酸亚铁0.08、硫酸镁0.3和磷酸氢二钾0.4。通过以上优化后的培养基培养的p.terodonis QZ-4,其褐藻胶裂解酶活力可达到148.24U/mL,与优化前相比,酶活提高了111.8%。
3.研究了不同的表面活性剂对菌体生长和产酶的作用,结果表明Tween 80对P. terodonisQZ-4产酶的诱导效果最好,在0.01-0.16%浓度范围内均能促进酶的产生,而Triton x-100、SDS对生长和产酶均有不同程度的抑制作用。
4.采用部分因子实验方法和响应面法对P. terodonis QZ-4发酵生产褐藻胶裂解酶的发酵条件进行优化,结果表明装液量等发酵条件对褐藻胶裂解酶活性有显著的影响；优化后的最佳发酵条件为：发酵温度25℃、转速160rpm、装液量为25mL、发酵时间为15h。理论最大产酶活力为229.40U/mL,实际发酵得到最大产酶活力为256.7U/mL,与优化前相比,酶活提高了73.2%,与起始酶活相比,提高了266.7%。
5.在10L发酵罐中研究了以褐藻胶为底物发酵产褐藻胶裂解酶的动力学,并建立了褐藻胶裂解酶的生成动力学模型,底物消耗动力学模型为和菌体生长动力学模型。
6.对褐藻胶裂解酶在PEG/(NH4)2SO4体系中的分配行为进行研究。发现在相同PEG浓度和硫酸铵浓度条件下,分子量范围在2000-6000的PEG,随着分子量的减小,体系的相比逐渐增大,但对酶和总蛋白在双水相体系的分配行为并无显著的影响；随着体系中酶液添加量的增加,分配系数均有所下降。当体系中PEG2000浓度从10%提高到25%时,相比增大,褐藻胶裂解酶的分配系数和萃取率分别从1.75和30.43%增加到10.97和88.76%。当体系中硫酸铵浓度从15%提高到30%时,相比减小,酶的分配系数和萃取率分别从从0.29和11.54%增加到5.14和70.72%。随着体系pH的增加,相比减小,酶的分配系数和回收率略有提高。
7.对纯化后褐藻胶裂解酶的理化性质及反应动力学进行了研究,结果表明：该酶具有同时降解M段和G段的能力,分子量为27 kDa,由L-B作图法求得最大反应速度Vmax=0.541U/mL,米氏常数Km=0.051mg/mL。最适作用pH值和温度分别为7.5和40℃,最适缓冲液为0.05mM Tris-HCl缓冲液,在偏酸或偏碱,以及40℃以上保存则活力快速下降；该酶为金属酶,1.0mol/L EDTA可完全抑制其活性,Mg2+、Na+、Fe3+、Mn2+均对酶活有促进作用,其中以Mg2+的促进作用最为显著,提高50%；Zn2+、Al3+、Fe2+、Cu2+均对酶的活力有抑制作用。
8.使用双水相萃取法制备的粗酶液,在35℃、100rpm恒温水浴条件下降解2%褐藻胶溶液,降解反应15～20h,沸水浴终止反应,通过离心和超滤两步分离,得到浓度为1.5～1.6%的酶解糖液。用GPC法分析糖液的分子量分布,以低分子量葡聚糖为标准品,推测制得的酶解寡糖混合液中各组分的聚合度在2～9之间。其中2-6聚合度的寡糖为主要成分,占总量的95.2%；酶解终产物为二糖和三塘,在酶解糖液中的含量分别为32.96%和31.45%。
9.制得的酶解糖液经稀释后,可以作为促种子萌发剂,分别采用种子萌发前浸种和种子萌发后喷洒的方式处理玉米和小麦种子。选用100ppm浓度左右的酶解糖液浸种处理后,玉米和小麦种子的种子生活力均得到提高,小麦的发芽势也得到提高,小麦发芽后的芽长和根数没有明显变化,但是根长有一定增加,根尖活力提高。若选用直接对芽和根喷洒酶解糖液的方式,适用浓度应大幅调低,可选择10ppm浓度左右的酶解糖液,对芽长和根的增加均有促进作用,并避免喷洒浓度500ppm以上的酶解糖液,否则对根的生长和根系活力均有抑制破坏作用。
Alginate lyases, also known as alginases or alginate depolymerases, catalyze the degradation of alginate byβ-elimination mechanism, forming 4-deoxy-L-erythro-hex-4-enopyranosyluronate at the new non-reducing terminus. The various alginate lyases were reported to be applied in broad fields, including analyzing the fine structure of alginate, preparing protoplasts of seaweed, preparing specific food products, degrading alginate polysaccharide build-up in the lungs of CF sufferers, et al. We reported here a marine bacterium Pseudoalteromonas tetrodonis QZ-4 over-producing alginate lyase. The culture composition and culture conditions were optimized. The processes of fermentation in 10L bioreactor were discussed. Then the purification and characterization of alginate lyase were discussed as well as the processes and mechanism. At last the influence of alginate-derived oligosaccharide was tested on the seed germization of maize and wheat.
The results were shown as below:
(1) A marine strain QZ-4 was screened from seawater sample and identified as Pseudoalteromonas tetrodonis based on phylogenetic characteristic (G+C,52.2%). The strain is a Gram-negative rod measuring 0.3μm by 1.0μm with a single polar flagellum, pigment negative. The clone was tiny(Φ1mm),round and white, with moist and smooth surface, when cultured in plate contained 2.5% NaCl medium, at 28℃for 1d.
(2) The effect of medium composition on alginate lyase production was investigated using surface response methodology. The concentrations of inorganic nitrogen and salts in culture medium, such as di-ammonium hydrogen phosphate, sodium chloride, ferrous sulfate, magnesium sulphate and potassium di-hydrogen phosphate, were changed to obtain the optimal model. The result showed that di-ammonium hydrogen phosphate, sodium chloride and magnesium sulphate had significant effects on alginate lyase production using a two-level fractional factorial design (FFD). The maximum yield of alginate lyase was attained in an optimized medium containing (g·L-1):Alginate sodium 5, (NH4)2HPO4 11.6, NaCl 32.9, FeSO4 0.03, MgSO4 0.08 and K2HPO4 0.4, using the surface response methodology and central composite design (CCD). The model prediction of alginate lyase yield at 145.45 U·ml-1 was experimentally verified.
(3) The effect of surfactant of the alginate lyase production by strain QZ-4 was studied. The results indicated that concentration at 0.01～0.16%(W/V) of Tween 80 could promote the strain to produce alginate lyase. Triton x-100 and SDS, however, inhibited the production of alginate lyase and the growth of strain QZ-4.
(4) The effect of culture condition on alginate lyase production was investigated using surface response methodology. Culture conditions as temperature, speed of rotation, loading volumn and fermentation time were studied. The result showed that loading volumn had significant effects on alginate lyase production using a two-level fractional factorial design. The maximum yield of alginate lyase was attained in an optimized culture condition:Temperature 25℃, speed of rotation 160rpm, loading volumn 25mL, time 15h, using the surface response methodology and central composite design (CCD). The model prediction of alginate lyase yield at 256.7 U·ml-1 was experimentally verified.
(5) During fermentation processes in 10L bioreactor, the model of alginate lyase production, kinetic models of cell growth and product formation were described repectively.
(6) The feasibility of alginate lyase purification from fermentation supernatant of strain QZ-4 with PEG-ammonium sulfate aqueous two-phase system was studied. PEG molecular weight(2000-6000), supernatant amount, pH, volumn of supernatant and concentration of PEG or ammonium sulfate had different effects on the partition of alginate lyase. A purification factor of 1.39 and 85% yield were reached.
(7) The alginate lyase was purified using 30-80% saturation ammonium sulphate precipitation, Sephadex G-100 and DEAE-Sepharose FF chromatographic techniques. The purified alginate lyase was detected as a single protein band upon SDS-PAGE, with a moleculr weight of 34.6KD. It showed optimum activity at a temperature of 40℃and a pH 7.5. Also it showed stability below 40℃and pH range 6.5～8.0. The enzyme was promoted by Mg2+、Na+、Fe3+、Mn2+ and inhibited by Zn2+、Al3+、Fe2+、Cu2+ and EDTA.
(8) The 2% alginate sodium solution was degraded by crude enzyme at 35℃for 15～20h, inactivated in boiling water. A mixture of oligosaccharide was obtained through centrifugation and ultrafiltration. The degree of degradation was analysed by HPGPC,using glucosans as standard. Degree of polymerization of different compositions was estimated as 2 to 6, amount to 95.2% and the end products of enzyme hydrolysis should be disaccharide and trisaccharide, amount to 32.96% and 31.45% respectively.
(9) The influence of alginate-derived oligosaccharide was tested on germination of maize and wheat seed by different way, soaking seed before germination or spraring after germination. Seed vitality, seed germination energy, shoot length, root length, catalase activity and root vitality were estimated to evaluate effects of oligosaccharide mixture with different concentration. Compared with the control, the seed vitality and germination energy of wheat showed increase at 100ppm soaking treatment. And the root vitality increased also. To treat germinated wheat seed with 10ppm oligosaccharide mixture by sparying, could promote root vitality. However treatment over 500 ppm could inhibite the normal germination of wheat seed.
1.在浙江舟山、山东青岛、西班牙等沿海地区采集样品,共采集海水、海藻、泥沙等样品58份,通过平板粗筛得到大量细菌、放线菌、酵母和霉菌,其中具有酶活性的细菌计483株、霉菌计1株；对这484株菌进行平板扩散筛选,结合摇瓶初筛和复筛手段,得到一株生长和产酶(70U/mL)较稳定的海洋细菌。该菌在2.5%NaCl浓度的分离筛选平板上,28℃培养1d,菌落为白色,圆形(Φ1mm),表面湿润光滑,不产色素,革兰氏染色阴性,菌体形态呈短杆状(0.2-0.4μmm×0.7-1μm),单端带鞭毛。通过16S rDNA鉴定,序列G+C含量为52.2%,BLAST程序比对,该菌株与Pseudoalteromonas tetrodonis相似性为97%,命名为Pseudoalteromonas terodonis QZ-4。
27采用部分因子实验方法和响应面法对P. terodonis QZ-4发酵生产褐藻胶裂解酶的培养基条件进行优化。首先明确了磷酸氢二铵、氯化钠和硫酸亚铁是影响褐藻胶裂解酶活性的主要因素,进而用最陡爬坡路径逼近最大响应区域,并采用中心组合设计及响应面分析确定了主要影响因子的最佳浓度,最终获得的优化培养基组成为(g/L)：褐藻酸钠5、磷酸氢二铵11.6、氯化钠32.9、硫酸亚铁0.08、硫酸镁0.3和磷酸氢二钾0.4。通过以上优化后的培养基培养的p.terodonis QZ-4,其褐藻胶裂解酶活力可达到148.24U/mL,与优化前相比,酶活提高了111.8%。
3.研究了不同的表面活性剂对菌体生长和产酶的作用,结果表明Tween 80对P. terodonisQZ-4产酶的诱导效果最好,在0.01-0.16%浓度范围内均能促进酶的产生,而Triton x-100、SDS对生长和产酶均有不同程度的抑制作用。
4.采用部分因子实验方法和响应面法对P. terodonis QZ-4发酵生产褐藻胶裂解酶的发酵条件进行优化,结果表明装液量等发酵条件对褐藻胶裂解酶活性有显著的影响；优化后的最佳发酵条件为：发酵温度25℃、转速160rpm、装液量为25mL、发酵时间为15h。理论最大产酶活力为229.40U/mL,实际发酵得到最大产酶活力为256.7U/mL,与优化前相比,酶活提高了73.2%,与起始酶活相比,提高了266.7%。
5.在10L发酵罐中研究了以褐藻胶为底物发酵产褐藻胶裂解酶的动力学,并建立了褐藻胶裂解酶的生成动力学模型,底物消耗动力学模型为和菌体生长动力学模型。
6.对褐藻胶裂解酶在PEG/(NH4)2SO4体系中的分配行为进行研究。发现在相同PEG浓度和硫酸铵浓度条件下,分子量范围在2000-6000的PEG,随着分子量的减小,体系的相比逐渐增大,但对酶和总蛋白在双水相体系的分配行为并无显著的影响；随着体系中酶液添加量的增加,分配系数均有所下降。当体系中PEG2000浓度从10%提高到25%时,相比增大,褐藻胶裂解酶的分配系数和萃取率分别从1.75和30.43%增加到10.97和88.76%。当体系中硫酸铵浓度从15%提高到30%时,相比减小,酶的分配系数和萃取率分别从从0.29和11.54%增加到5.14和70.72%。随着体系pH的增加,相比减小,酶的分配系数和回收率略有提高。
7.对纯化后褐藻胶裂解酶的理化性质及反应动力学进行了研究,结果表明：该酶具有同时降解M段和G段的能力,分子量为27 kDa,由L-B作图法求得最大反应速度Vmax=0.541U/mL,米氏常数Km=0.051mg/mL。最适作用pH值和温度分别为7.5和40℃,最适缓冲液为0.05mM Tris-HCl缓冲液,在偏酸或偏碱,以及40℃以上保存则活力快速下降；该酶为金属酶,1.0mol/L EDTA可完全抑制其活性,Mg2+、Na+、Fe3+、Mn2+均对酶活有促进作用,其中以Mg2+的促进作用最为显著,提高50%；Zn2+、Al3+、Fe2+、Cu2+均对酶的活力有抑制作用。
8.使用双水相萃取法制备的粗酶液,在35℃、100rpm恒温水浴条件下降解2%褐藻胶溶液,降解反应15～20h,沸水浴终止反应,通过离心和超滤两步分离,得到浓度为1.5～1.6%的酶解糖液。用GPC法分析糖液的分子量分布,以低分子量葡聚糖为标准品,推测制得的酶解寡糖混合液中各组分的聚合度在2～9之间。其中2-6聚合度的寡糖为主要成分,占总量的95.2%；酶解终产物为二糖和三塘,在酶解糖液中的含量分别为32.96%和31.45%。
9.制得的酶解糖液经稀释后,可以作为促种子萌发剂,分别采用种子萌发前浸种和种子萌发后喷洒的方式处理玉米和小麦种子。选用100ppm浓度左右的酶解糖液浸种处理后,玉米和小麦种子的种子生活力均得到提高,小麦的发芽势也得到提高,小麦发芽后的芽长和根数没有明显变化,但是根长有一定增加,根尖活力提高。若选用直接对芽和根喷洒酶解糖液的方式,适用浓度应大幅调低,可选择10ppm浓度左右的酶解糖液,对芽长和根的增加均有促进作用,并避免喷洒浓度500ppm以上的酶解糖液,否则对根的生长和根系活力均有抑制破坏作用。
Alginate lyases, also known as alginases or alginate depolymerases, catalyze the degradation of alginate byβ-elimination mechanism, forming 4-deoxy-L-erythro-hex-4-enopyranosyluronate at the new non-reducing terminus. The various alginate lyases were reported to be applied in broad fields, including analyzing the fine structure of alginate, preparing protoplasts of seaweed, preparing specific food products, degrading alginate polysaccharide build-up in the lungs of CF sufferers, et al. We reported here a marine bacterium Pseudoalteromonas tetrodonis QZ-4 over-producing alginate lyase. The culture composition and culture conditions were optimized. The processes of fermentation in 10L bioreactor were discussed. Then the purification and characterization of alginate lyase were discussed as well as the processes and mechanism. At last the influence of alginate-derived oligosaccharide was tested on the seed germization of maize and wheat.
The results were shown as below:
(1) A marine strain QZ-4 was screened from seawater sample and identified as Pseudoalteromonas tetrodonis based on phylogenetic characteristic (G+C,52.2%). The strain is a Gram-negative rod measuring 0.3μm by 1.0μm with a single polar flagellum, pigment negative. The clone was tiny(Φ1mm),round and white, with moist and smooth surface, when cultured in plate contained 2.5% NaCl medium, at 28℃for 1d.
(2) The effect of medium composition on alginate lyase production was investigated using surface response methodology. The concentrations of inorganic nitrogen and salts in culture medium, such as di-ammonium hydrogen phosphate, sodium chloride, ferrous sulfate, magnesium sulphate and potassium di-hydrogen phosphate, were changed to obtain the optimal model. The result showed that di-ammonium hydrogen phosphate, sodium chloride and magnesium sulphate had significant effects on alginate lyase production using a two-level fractional factorial design (FFD). The maximum yield of alginate lyase was attained in an optimized medium containing (g·L-1):Alginate sodium 5, (NH4)2HPO4 11.6, NaCl 32.9, FeSO4 0.03, MgSO4 0.08 and K2HPO4 0.4, using the surface response methodology and central composite design (CCD). The model prediction of alginate lyase yield at 145.45 U·ml-1 was experimentally verified.
(3) The effect of surfactant of the alginate lyase production by strain QZ-4 was studied. The results indicated that concentration at 0.01～0.16%(W/V) of Tween 80 could promote the strain to produce alginate lyase. Triton x-100 and SDS, however, inhibited the production of alginate lyase and the growth of strain QZ-4.
(4) The effect of culture condition on alginate lyase production was investigated using surface response methodology. Culture conditions as temperature, speed of rotation, loading volumn and fermentation time were studied. The result showed that loading volumn had significant effects on alginate lyase production using a two-level fractional factorial design. The maximum yield of alginate lyase was attained in an optimized culture condition:Temperature 25℃, speed of rotation 160rpm, loading volumn 25mL, time 15h, using the surface response methodology and central composite design (CCD). The model prediction of alginate lyase yield at 256.7 U·ml-1 was experimentally verified.
(5) During fermentation processes in 10L bioreactor, the model of alginate lyase production, kinetic models of cell growth and product formation were described repectively.
(6) The feasibility of alginate lyase purification from fermentation supernatant of strain QZ-4 with PEG-ammonium sulfate aqueous two-phase system was studied. PEG molecular weight(2000-6000), supernatant amount, pH, volumn of supernatant and concentration of PEG or ammonium sulfate had different effects on the partition of alginate lyase. A purification factor of 1.39 and 85% yield were reached.
(7) The alginate lyase was purified using 30-80% saturation ammonium sulphate precipitation, Sephadex G-100 and DEAE-Sepharose FF chromatographic techniques. The purified alginate lyase was detected as a single protein band upon SDS-PAGE, with a moleculr weight of 34.6KD. It showed optimum activity at a temperature of 40℃and a pH 7.5. Also it showed stability below 40℃and pH range 6.5～8.0. The enzyme was promoted by Mg2+、Na+、Fe3+、Mn2+ and inhibited by Zn2+、Al3+、Fe2+、Cu2+ and EDTA.
(8) The 2% alginate sodium solution was degraded by crude enzyme at 35℃for 15～20h, inactivated in boiling water. A mixture of oligosaccharide was obtained through centrifugation and ultrafiltration. The degree of degradation was analysed by HPGPC,using glucosans as standard. Degree of polymerization of different compositions was estimated as 2 to 6, amount to 95.2% and the end products of enzyme hydrolysis should be disaccharide and trisaccharide, amount to 32.96% and 31.45% respectively.
(9) The influence of alginate-derived oligosaccharide was tested on germination of maize and wheat seed by different way, soaking seed before germination or spraring after germination. Seed vitality, seed germination energy, shoot length, root length, catalase activity and root vitality were estimated to evaluate effects of oligosaccharide mixture with different concentration. Compared with the control, the seed vitality and germination energy of wheat showed increase at 100ppm soaking treatment. And the root vitality increased also. To treat germinated wheat seed with 10ppm oligosaccharide mixture by sparying, could promote root vitality. However treatment over 500 ppm could inhibite the normal germination of wheat seed.
引文
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