【摘要】：聚乙烯醇(polyvinyl alcohol)簡稱PVA,是一種高分子聚合物,它具有良好的水溶性,并且可以通過生物法來進行降解。由于它的可生物降解性,近些年來這種潛力受到了廣泛關(guān)注。PVA具有熱穩(wěn)定性、成膜性、乳化性、高粘度等許多優(yōu)良的特性,我國每年都要消耗大量的PVA。巨大的使用量下,PVA廢水的排放帶來了環(huán)境問題。盡管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。因此本論文從PVA微生物法降解和PVA酶法降解這兩方面研究PVA的生物降解,提高PVA生物降解速率。主要研究結(jié)果如下:(1)在無錫太平洋集團富含PVA的地點取樣,富集、傳代馴化后篩選到一個可以在48 h內(nèi)完全降解1 g·L~(-1) PVA的混菌體系BQ-2,從中分離純化出一株P(guān)VA降解能力最強的單菌F2,經(jīng)鑒定F2屬于鞘脂單胞菌屬(Sphingopyxis sp.)。對培養(yǎng)基中酵母粉添加量進行優(yōu)化,進一步提高F2降解PVA的性能。F2在添加1 g·L~(-1)酵母粉的培養(yǎng)基中,生長狀況良好,培養(yǎng)120 h,對1 g·L~(-1) PVA的降解率達到90%以上。相同條件下,相比在無機氮培養(yǎng)基中約50%的降解率,F2的降解能力提高了80%以上。在F2的細胞破碎上清液中檢測到了聚乙烯醇脫氫酶(PVADH)酶活和氧化型PVA水解酶(OPH)酶活。(2)以F2為出發(fā)菌株,進行等離子誘變,以獲得PVA降解能力更強的菌株�；谥滤缆�,確定最佳誘變時間為12 s。通過高通量篩選方法對經(jīng)過誘變處理后的菌株進行篩選,來獲得PVA降解能力提高的正向突變株。經(jīng)過搖瓶復篩,72 h后,得到一株P(guān)VA降解率比出發(fā)菌株F2提高了53%的突變株SM2。經(jīng)過遺傳穩(wěn)定性分析發(fā)現(xiàn),SM2連續(xù)傳10代,每代PVA降解率的變化幅度均在10%以內(nèi),表明誘變菌株SM2具有良好的遺傳穩(wěn)定性。(3)以F2的基因組DNA為pvadh和oph的基因來源,分別實現(xiàn)PVADH和OPH高效表達。將密碼子優(yōu)化后的pvadh基因片段插入表達載體pPIC9K的多克隆位點,得到pPIC9K/pvadh,經(jīng)Sal I線性化后電轉(zhuǎn)化工程菌P.pastoris GS115,選擇在G418濃度為3 mg·mL~(-1)的YPD平板上篩選到的P.pastoris GS115/pPIC9K/pvadh進行搖瓶發(fā)酵,經(jīng)甲醇誘導120 h,PVADH酶活最高達到約523 U·mL~(-1);將oph基因片段插入表達載體pET-20b(+)的多克隆位點,得到pET-20b(+)/oph,轉(zhuǎn)化工程菌E.coli BL21(DE3),將重組菌株E.coli BL21(DE3)/pET-20b(+)/oph進行搖瓶發(fā)酵,誘導120 h,OPH酶活最高達到約20.6 U·mL~(-1)。(4)探究PVA的酶法生物降解,對PVADH和OPH雙酶降解PVA的工藝條件進行優(yōu)化。研究了PVADH酶量、OPH酶量、反應(yīng)溫度、pH、Ca2+和PVA濃度這6個單因素對PVA降解率的影響。通過單因素實驗,選擇三個顯著的因素,即PVADH酶量、pH和反應(yīng)溫度進行最陡爬坡實驗、Box-Behnken實驗以及響應(yīng)面分析,最終得到了優(yōu)化的PVA雙酶降解工藝條件:PVADH 123 U·m L~(-1)、pH 7.7、反應(yīng)溫度41℃、OPH 12 U·m L~(-1)、PVA 1 g·L~(-1)、Ca2+濃度1 mmol·L~(-1)、PQQ濃度6μmol·L~(-1)。在優(yōu)化條件下,反應(yīng)進行1 h,PVA降解率為34.07%;反應(yīng)4 h,PVA降解率達到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，

本文編號：2399738