聚乙烯醇降解菌的筛选及其关键酶的应用研究
发布时间：2019-01-03 18:33
【摘要】：聚乙烯醇(polyvinyl alcohol)简称PVA,是一种高分子聚合物,它具有良好的水溶性,并且可以通过生物法来进行降解。由于它的可生物降解性,近些年来这种潜力受到了广泛关注。PVA具有热稳定性、成膜性、乳化性、高粘度等许多优良的特性,我国每年都要消耗大量的PVA。巨大的使用量下,PVA废水的排放带来了环境问题。尽管PVA可生物降解,但存在很大的局限性,其生物降解速度慢,降解效率低。因此本论文从PVA微生物法降解和PVA酶法降解这两方面研究PVA的生物降解,提高PVA生物降解速率。主要研究结果如下:(1)在无锡太平洋集团富含PVA的地点取样,富集、传代驯化后筛选到一个可以在48 h内完全降解1 g·L~(-1) PVA的混菌体系BQ-2,从中分离纯化出一株PVA降解能力最强的单菌F2,经鉴定F2属于鞘脂单胞菌属(Sphingopyxis sp.)。对培养基中酵母粉添加量进行优化,进一步提高F2降解PVA的性能。F2在添加1 g·L~(-1)酵母粉的培养基中,生长状况良好,培养120 h,对1 g·L~(-1) PVA的降解率达到90%以上。相同条件下,相比在无机氮培养基中约50%的降解率,F2的降解能力提高了80%以上。在F2的细胞破碎上清液中检测到了聚乙烯醇脱氢酶(PVADH)酶活和氧化型PVA水解酶(OPH)酶活。(2)以F2为出发菌株,进行等离子诱变,以获得PVA降解能力更强的菌株。基于致死率,确定最佳诱变时间为12 s。通过高通量筛选方法对经过诱变处理后的菌株进行筛选,来获得PVA降解能力提高的正向突变株。经过摇瓶复筛,72 h后,得到一株PVA降解率比出发菌株F2提高了53%的突变株SM2。经过遗传稳定性分析发现,SM2连续传10代,每代PVA降解率的变化幅度均在10%以内,表明诱变菌株SM2具有良好的遗传稳定性。(3)以F2的基因组DNA为pvadh和oph的基因来源,分别实现PVADH和OPH高效表达。将密码子优化后的pvadh基因片段插入表达载体pPIC9K的多克隆位点,得到pPIC9K/pvadh,经Sal I线性化后电转化工程菌P.pastoris GS115,选择在G418浓度为3 mg·mL~(-1)的YPD平板上筛选到的P.pastoris GS115/pPIC9K/pvadh进行摇瓶发酵,经甲醇诱导120 h,PVADH酶活最高达到约523 U·mL~(-1);将oph基因片段插入表达载体pET-20b(+)的多克隆位点,得到pET-20b(+)/oph,转化工程菌E.coli BL21(DE3),将重组菌株E.coli BL21(DE3)/pET-20b(+)/oph进行摇瓶发酵,诱导120 h,OPH酶活最高达到约20.6 U·mL~(-1)。(4)探究PVA的酶法生物降解,对PVADH和OPH双酶降解PVA的工艺条件进行优化。研究了PVADH酶量、OPH酶量、反应温度、pH、Ca2+和PVA浓度这6个单因素对PVA降解率的影响。通过单因素实验,选择三个显著的因素,即PVADH酶量、pH和反应温度进行最陡爬坡实验、Box-Behnken实验以及响应面分析,最终得到了优化的PVA双酶降解工艺条件:PVADH 123 U·m L~(-1)、pH 7.7、反应温度41℃、OPH 12 U·m L~(-1)、PVA 1 g·L~(-1)、Ca2+浓度1 mmol·L~(-1)、PQQ浓度6μmol·L~(-1)。在优化条件下,反应进行1 h,PVA降解率为34.07%;反应4 h,PVA降解率达到95%以上。
[Abstract]:Polyvinyl alcohol (polyvinyl alcohol) is a kind of polymer, which has good water solubility and can be degraded by biological method. Due to its biodegradability, this potential has attracted wide attention in recent years. PVA has many excellent properties, such as thermal stability, film-forming, emulsification, high viscosity and so on, and a large amount of PVA. is consumed every year in China. Under the huge usage, the discharge of PVA wastewater brings environmental problems. Although PVA is biodegradable, it has many limitations, such as slow biodegradation rate and low degradation efficiency. In this paper, the biodegradation of PVA was studied from the aspects of microbial degradation of PVA and enzymatic degradation of PVA, and the biodegradation rate of PVA was improved. The main results are as follows: (1) the PVA rich sites in Wuxi Pacific Group were sampled, enriched, subcultured and domesticated, and a mixed bacteria system, BQ-2, which could completely degrade 1 g L ~ (-1) PVA within 48 h, was selected. A strain of F _ 2, which has the strongest ability to degrade PVA, was isolated and purified, and F2 was identified as belonging to (Sphingopyxis sp.). Of sphingomonas. The addition of yeast powder to the culture medium was optimized to further improve the ability of F2 to degrade PVA. F2 in the medium supplemented with 1 g L ~ (-1) yeast powder grew well and cultured for 120h. The degradation rate of 1 g L ~ (-1) PVA was over 90%. Under the same conditions, compared with the degradation rate of about 50% in inorganic nitrogen medium, the degradation ability of F2 was increased by more than 80%. The activity of polyvinyl alcohol dehydrogenase (PVADH) and oxidized PVA hydrolase (OPH) were detected in the supernatant of F _ 2 cell fragmentation. (2) using F2 as the starting strain, plasma mutagenesis was carried out to obtain the strain with stronger PVA degradation ability. Based on the mortality, the optimal mutagenic time was 12 s. High throughput screening method was used to screen the mutagenic strains to obtain the positive mutants with improved PVA degradation ability. After shaking flask screening and 72 h later, a mutant strain SM2. with a 53% higher degradation rate of PVA than the original strain F2 was obtained. Genetic stability analysis showed that the degradation rate of PVA was within 10% in 10 generations of SM2, indicating that the mutagenesis strain SM2 had good genetic stability. (3) Genomic DNA of F2 was used as the gene source of pvadh and oph. PVADH and OPH are expressed efficiently, respectively. The codon optimized pvadh gene fragment was inserted into the polyclonal site of the expression vector pPIC9K, and the pPIC9K/pvadh, was linearized by Sal I to be electrotransformed into the engineering bacterium P.pastoris GS115,. The P.pastoris GS115/pPIC9K/pvadh selected on the YPD plate with G418 concentration of 3 mg mL~ (-1) was selected for shaking flask fermentation. The maximum activity of PVADH was about 523 U mL~ (-1) induced by methanol. The oph gene fragment was inserted into the polyclonal site of the expression vector pET-20b () to obtain pET-20b () / oph, transformed engineering bacterium E.coli BL21 (DE3). The recombinant strain E.coli BL21 (DE3) / pET-20b () / oph was flask fermented. The maximum activity of OPH reached 20.6 U mL~ (-1). (4) at 120h after induction to explore the enzymatic biodegradation of PVA, and to optimize the process conditions for the degradation of PVA by PVADH and OPH. The effects of PVADH enzyme amount, OPH enzyme amount, reaction temperature and concentration of pH,Ca2 and PVA on the degradation rate of PVA were studied. Through the single factor experiment, the steepest climbing experiment, the Box-Behnken experiment and the response surface analysis were carried out by selecting three significant factors, namely, the amount of PVADH enzyme, pH and reaction temperature. The optimum conditions of PVA degradation were obtained as follows: PVADH 123U mL ~ (-1), pH, reaction temperature 41 鈩，
本文编号：2399738
[Abstract]:Polyvinyl alcohol (polyvinyl alcohol) is a kind of polymer, which has good water solubility and can be degraded by biological method. Due to its biodegradability, this potential has attracted wide attention in recent years. PVA has many excellent properties, such as thermal stability, film-forming, emulsification, high viscosity and so on, and a large amount of PVA. is consumed every year in China. Under the huge usage, the discharge of PVA wastewater brings environmental problems. Although PVA is biodegradable, it has many limitations, such as slow biodegradation rate and low degradation efficiency. In this paper, the biodegradation of PVA was studied from the aspects of microbial degradation of PVA and enzymatic degradation of PVA, and the biodegradation rate of PVA was improved. The main results are as follows: (1) the PVA rich sites in Wuxi Pacific Group were sampled, enriched, subcultured and domesticated, and a mixed bacteria system, BQ-2, which could completely degrade 1 g L ~ (-1) PVA within 48 h, was selected. A strain of F _ 2, which has the strongest ability to degrade PVA, was isolated and purified, and F2 was identified as belonging to (Sphingopyxis sp.). Of sphingomonas. The addition of yeast powder to the culture medium was optimized to further improve the ability of F2 to degrade PVA. F2 in the medium supplemented with 1 g L ~ (-1) yeast powder grew well and cultured for 120h. The degradation rate of 1 g L ~ (-1) PVA was over 90%. Under the same conditions, compared with the degradation rate of about 50% in inorganic nitrogen medium, the degradation ability of F2 was increased by more than 80%. The activity of polyvinyl alcohol dehydrogenase (PVADH) and oxidized PVA hydrolase (OPH) were detected in the supernatant of F _ 2 cell fragmentation. (2) using F2 as the starting strain, plasma mutagenesis was carried out to obtain the strain with stronger PVA degradation ability. Based on the mortality, the optimal mutagenic time was 12 s. High throughput screening method was used to screen the mutagenic strains to obtain the positive mutants with improved PVA degradation ability. After shaking flask screening and 72 h later, a mutant strain SM2. with a 53% higher degradation rate of PVA than the original strain F2 was obtained. Genetic stability analysis showed that the degradation rate of PVA was within 10% in 10 generations of SM2, indicating that the mutagenesis strain SM2 had good genetic stability. (3) Genomic DNA of F2 was used as the gene source of pvadh and oph. PVADH and OPH are expressed efficiently, respectively. The codon optimized pvadh gene fragment was inserted into the polyclonal site of the expression vector pPIC9K, and the pPIC9K/pvadh, was linearized by Sal I to be electrotransformed into the engineering bacterium P.pastoris GS115,. The P.pastoris GS115/pPIC9K/pvadh selected on the YPD plate with G418 concentration of 3 mg mL~ (-1) was selected for shaking flask fermentation. The maximum activity of PVADH was about 523 U mL~ (-1) induced by methanol. The oph gene fragment was inserted into the polyclonal site of the expression vector pET-20b () to obtain pET-20b () / oph, transformed engineering bacterium E.coli BL21 (DE3). The recombinant strain E.coli BL21 (DE3) / pET-20b () / oph was flask fermented. The maximum activity of OPH reached 20.6 U mL~ (-1). (4) at 120h after induction to explore the enzymatic biodegradation of PVA, and to optimize the process conditions for the degradation of PVA by PVADH and OPH. The effects of PVADH enzyme amount, OPH enzyme amount, reaction temperature and concentration of pH,Ca2 and PVA on the degradation rate of PVA were studied. Through the single factor experiment, the steepest climbing experiment, the Box-Behnken experiment and the response surface analysis were carried out by selecting three significant factors, namely, the amount of PVADH enzyme, pH and reaction temperature. The optimum conditions of PVA degradation were obtained as follows: PVADH 123U mL ~ (-1), pH, reaction temperature 41 鈩，
本文编号：2399738
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