盾壳霉胞外蛋白酶特性、分离纯化及其生防作用评估
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摘要
盾壳霉是防治核盘菌的重要生防菌。在盾壳霉寄生核盘菌的过程中会分泌多种胞外酶,包括葡聚糖酶、几丁质酶和蛋白酶。前人已对盾壳霉产生葡聚糖酶和几丁质酶及其生防作用进行了许多研究,但对盾壳霉产生蛋白酶及其生防作用尚未见报道。为了明确蛋白酶在盾壳霉寄生核盘菌中的作用,本课题开展了对盾壳霉产生胞外蛋白酶的条件、盾壳霉蛋白酶基本酶学性质、蛋白酶分离纯化、以及蛋白酶粗提物对核盘菌菌丝和原生质体的作用效果等方面进行了研究。
比较了马铃薯葡萄糖培养液(PDB)、马铃薯培养液(PB)、核盘菌菌核提取物培养液(Ss)、马铃薯菌核提取物培养液(PBSs)、马铃薯葡萄糖菌核提取物培养液(PDBSs)5种培养基对盾壳霉胞外蛋白酶产量影响。结果发现Ss培养液适合盾壳霉产生蛋白酶。结果还表明：葡萄糖不利于盾壳霉产生蛋白酶。
采用福林酚法对盾壳霉胞外蛋白酶活性进行了定量测定,并研究了影响盾壳霉蛋白酶活性的因子。结果表明：盾壳霉蛋白酶活性最适温度为60℃,最适pH值为7。在40℃以下保存,蛋白酶稳定；一些金属离子,包括Na+、K+、Li+、Mg2+、Zn2+、Ca2+、Cu2+和Nn2+等(5mM)对盾壳霉蛋白酶酶活没有影响；而Fe2+(5 mM)对盾壳霉蛋白酶活性发挥具有明显的促进作用；当Fe2+浓度达到250 mM时,盾壳霉蛋白酶活性发挥受到显著抑制。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)非常敏感,说明盾壳霉产生的蛋白酶可能是一种丝氨酸蛋白酶。
经过硫酸铵沉淀、透析后的样品,通过Q sepharose Fast Flow阴离子交换层析后,纯度有所提高,但仍未达到电泳纯,收集管中可以检测到蛋白酶的活性,为0.3U/ml,经过超滤,通过HiPrep 16/60 sephocryl s-100 High Resolution层析后,虽达到电泳纯,但未检测到蛋白酶活性,问题的原因还有待进一步探讨。
经过Q sepharose Fast Flow层析收集液,检测到蛋白酶活性的收集管,处理核盘菌菌丝和原生质体,可以降解核盘菌的菌丝,也可以降解核盘菌的原生质体,说明盾壳霉产生蛋白酶在其重寄生过程中扮演重要角色。
Coniothyrium minitans is an important biocontrol agent of Sclerotinia sclerotiorum. During the process that C. minitans parasited SI sclerotiorum, C. minitans would secret many extracellular enzymes including glucanase, chitinase and protease. The biocontrol function of glucanase and chitinase during interaction between C. minitans and S. sclerotiorum had been well studied in previous researches, however, the bicocontrol function of protease remain to be determined. In order to clarify the role of protease during interaction between the two organisms, A study was carried out on the protease production conditions, protease properties, protease purification and the effect of the crude extraction of protease on S.sclerotiorum mycelium and protoplasts.
Five media, namely potato dextrose broth (PDB), potato broth (PB), S. sclerotiorum extracts (Ss), potato broth S. sclerotiorum extracts (PBSs) and potato dextrose broth S. sclerotiorum extracts (PDBSs) were used for comparing the yield of the protease. Results showed that the medium Ss was the most suitable medium for protease production by C. minitans. In addition, the result also showed that glucose inhibited the production of protease.
The activity of protease produced by C. minitans were measured quantitatively by Folin phenol method and the factors affected the protease activity were also determined. The results were:the optimum temperature of the crude protease was 60℃, the optimum pH was 7, and the protease was stable bellow 40℃.5 mM Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Cu2+, Mn2+ had no effect on protease activity,5 mM Fe2+ could enhance the protease activity, whereas 250mM would inhibit the the protease activity. The protease was highly sensitive to PMSF (phenylmethyl sulfony fluoride) indicating it belonged to the serine protease family.
The proteinase was purified by ammonium sulfate, dialysis, Q sepharose Fast Flow chromatography. Its purity was checked to be about four bands by SDS-PAGE. The protease activity could be detected. Then, using ultrafiltration and HiPrep 16/60 sephocryl s-100 High Resolution chromatography, it was checked to be a single band by SDS-PAGE, but the protease activity could not be detected. This problem needed to be resolved in further study.
After a Q sepharose Fast Flow chromatography, S. sclerotiorum mycelium and protoplasts we're treated with the colletion that showed protease activity, the result showed that the colletion could degrade S. sclerotiorum mycelium and protoplasts. All the rusults indicate that the protease produced by C. minitans may play an important role in the infection of S. sclerotiorum by C. minitans.
比较了马铃薯葡萄糖培养液(PDB)、马铃薯培养液(PB)、核盘菌菌核提取物培养液(Ss)、马铃薯菌核提取物培养液(PBSs)、马铃薯葡萄糖菌核提取物培养液(PDBSs)5种培养基对盾壳霉胞外蛋白酶产量影响。结果发现Ss培养液适合盾壳霉产生蛋白酶。结果还表明：葡萄糖不利于盾壳霉产生蛋白酶。
采用福林酚法对盾壳霉胞外蛋白酶活性进行了定量测定,并研究了影响盾壳霉蛋白酶活性的因子。结果表明：盾壳霉蛋白酶活性最适温度为60℃,最适pH值为7。在40℃以下保存,蛋白酶稳定；一些金属离子,包括Na+、K+、Li+、Mg2+、Zn2+、Ca2+、Cu2+和Nn2+等(5mM)对盾壳霉蛋白酶酶活没有影响；而Fe2+(5 mM)对盾壳霉蛋白酶活性发挥具有明显的促进作用；当Fe2+浓度达到250 mM时,盾壳霉蛋白酶活性发挥受到显著抑制。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)非常敏感,说明盾壳霉产生的蛋白酶可能是一种丝氨酸蛋白酶。
经过硫酸铵沉淀、透析后的样品,通过Q sepharose Fast Flow阴离子交换层析后,纯度有所提高,但仍未达到电泳纯,收集管中可以检测到蛋白酶的活性,为0.3U/ml,经过超滤,通过HiPrep 16/60 sephocryl s-100 High Resolution层析后,虽达到电泳纯,但未检测到蛋白酶活性,问题的原因还有待进一步探讨。
经过Q sepharose Fast Flow层析收集液,检测到蛋白酶活性的收集管,处理核盘菌菌丝和原生质体,可以降解核盘菌的菌丝,也可以降解核盘菌的原生质体,说明盾壳霉产生蛋白酶在其重寄生过程中扮演重要角色。
Coniothyrium minitans is an important biocontrol agent of Sclerotinia sclerotiorum. During the process that C. minitans parasited SI sclerotiorum, C. minitans would secret many extracellular enzymes including glucanase, chitinase and protease. The biocontrol function of glucanase and chitinase during interaction between C. minitans and S. sclerotiorum had been well studied in previous researches, however, the bicocontrol function of protease remain to be determined. In order to clarify the role of protease during interaction between the two organisms, A study was carried out on the protease production conditions, protease properties, protease purification and the effect of the crude extraction of protease on S.sclerotiorum mycelium and protoplasts.
Five media, namely potato dextrose broth (PDB), potato broth (PB), S. sclerotiorum extracts (Ss), potato broth S. sclerotiorum extracts (PBSs) and potato dextrose broth S. sclerotiorum extracts (PDBSs) were used for comparing the yield of the protease. Results showed that the medium Ss was the most suitable medium for protease production by C. minitans. In addition, the result also showed that glucose inhibited the production of protease.
The activity of protease produced by C. minitans were measured quantitatively by Folin phenol method and the factors affected the protease activity were also determined. The results were:the optimum temperature of the crude protease was 60℃, the optimum pH was 7, and the protease was stable bellow 40℃.5 mM Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Cu2+, Mn2+ had no effect on protease activity,5 mM Fe2+ could enhance the protease activity, whereas 250mM would inhibit the the protease activity. The protease was highly sensitive to PMSF (phenylmethyl sulfony fluoride) indicating it belonged to the serine protease family.
The proteinase was purified by ammonium sulfate, dialysis, Q sepharose Fast Flow chromatography. Its purity was checked to be about four bands by SDS-PAGE. The protease activity could be detected. Then, using ultrafiltration and HiPrep 16/60 sephocryl s-100 High Resolution chromatography, it was checked to be a single band by SDS-PAGE, but the protease activity could not be detected. This problem needed to be resolved in further study.
After a Q sepharose Fast Flow chromatography, S. sclerotiorum mycelium and protoplasts we're treated with the colletion that showed protease activity, the result showed that the colletion could degrade S. sclerotiorum mycelium and protoplasts. All the rusults indicate that the protease produced by C. minitans may play an important role in the infection of S. sclerotiorum by C. minitans.
引文
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