粪便微生物宏基因组来源的热稳定性邻苯二酚1,2-双加氧酶异源表达及酶学性质

【目的】克隆倭蜂猴粪便微生物宏基因组的邻苯二酚1,2-双加氧酶基因cat PLCgl,并对该酶进行异源表达及酶学特性研究。【方法】利用宏基因组高通量测序技术获得cat PLCgl,并对其氨基酸序列进行分析。将cat PLCgl重组到载体p EASY-E2中并转化到大肠杆菌BL21(DE3)中异源表达,研究其酶学性质。【结果】cat PLCgl全长852 bp,G+C含量48%,编码283个氨基酸,理论分子量为33.56 k D。重组Cat PLCgl酶学性质分析显示最适作用p H为7.0,其中在p H 7.0–10.0范围内处理1 h后,酶活剩余90%以上;最适作用温度为40°C,在25°C和40°C条件下稳定性较好,耐受210 h酶活性几乎不变。重组酶在最适条件下的动力学参数K_m、V_(max)和k_(cat)分别为24.9μmol/L、8.3 mmol/(min·g)和13.7 s~(-1);Fe~(2+)、Hg~(2+)、Cu~(2+)、Triton X-100、SDS、Ag+强烈抑制该酶活性,而其它金属离子及有机试剂影响较小。【结论】从倭蜂猴粪便微生物宏基因组中克隆得到邻苯二酚1,2-双加氧酶基因cat PLCgl,并对重组Cat PLCgl酶学性质进行研究,该酶具有较好的热稳定性和耐碱性,在降解环境中的邻苯二酚和生产顺,顺-己二烯二酸方面具有应用潜力。     

邻苯二酚-1,2-双加氧酶的催化性质

邻苯二酚-1,2-双加氧酶能催化邻苯二酚的两个羟基之间裂解,生成顺,顺-己二烯二酸,加水很容易转化为尼龙-6,6的原料己二酸。此外,它有极易起化学反应的共轭双键和羧基,可成为新功能树脂的原料。1955年发现邻苯二酚-1,2-双加氧酶,此后进行了大量研究工作,但是直到近几年由于合成顺,顺-己二烯二酸的需要,才引起人们的高度重视。我们对胞外酶产生菌的发酵条件及其所产的邻苯二酚-1,2-双加氧酶性质进行了研究,为工业规模的生产应用作了准备。     

产胞外邻苯二酚1，2-双加氧酶的菌种筛选和发酵条件的研究

从112株细菌中筛选出两株产胞外邻苯二酚1,2一双加氧酶的假单胞菌(Pseubomonassp.)。 进行了该菌产酶的发酵条件试验。产酶的最适温度为30℃,最适起始pH为6．8—7-0。葡萄糖、麦芽糖和甘油对产酶有明显的抑制作用,苯甲酸钠对产酶有促进作用。氨态氮对菌体生长和产酶是必需的。琥珀酸钠是酶形成的有教诱导物。采用0.15％苯甲酸钠培养基(pH6.8—7.0),于30℃振荡培养72h,每毫升发酵液酶活力可达10单位。     

邻苯二酚1,2-双加氧酶的结构、功能及同工酶现象研究进展

邻苯二酚是芳香族化合物多条生物降解途径中共有的一种重要的中间产物,根据开环方式的不同,可分为邻位降解途径和间位降解途径,其中邻位降解途径中的关键酶是邻苯二酚1,2-双加氧酶。本文主要综述了邻苯二酚1,2-双加氧酶的结构、酶学性质,以及它在芳香烃降解菌中存在的同工酶现象及其功能研究进展。     

邻苯二酚2,3-双加氧酶的结构和功能研究进展

邻苯二酚是所有芳香族化合物降解过程中的重要的中间产物,其降解有邻位和间位裂解两条裂解途径,分别由邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)催化裂解。本综述简要介绍了邻苯二酚2,3-双加氧酶的结构和功能的研究进展。     

L-异亮氨酸和甘氨酸对大肠杆菌表达与分泌邻苯二酚2,3-双加氧酶的作用

证实了甘氨酸与L-异亮氨酸对大肠杆菌表达邻苯二酚2,3-双加氧酶(CatO_2ase)的促进作用和甘氨酸促使该酶分泌至胞外培养基中的作用.产酶量高低和分泌量多少与培养基种类、甘氨酸和L-异亮氨酸的浓度以及培养时间等因素有关.在甘氨酸存在的情况下,胞壁对溶菌酶的敏感性有所增加,超微形态似有变化,还存在其他物质的伴随分泌,故甘氨酸可能是引起细胞壁结构的改变而导致邻苯二酚2,3-双加氧酶等胞内容物被动分泌至胞外.     

L-异亮氨酸和甘氨酸对大肠杆菌表达与分泌邻苯二酚2,3-双加氧酶的作用

证实了甘氨酸与L-异亮氨酸对大肠杆菌表达邻苯二酚2,3-双加氧酶(CatO_2ase)的促进作用和甘氨酸促使该酶分泌至胞外培养基中的作用.产酶量高低和分泌量多少与培养基种类、甘氨酸和L-异亮氨酸的浓度以及培养时间等因素有关.在甘氨酸存在的情况下,胞壁对溶菌酶的敏感性有所增加,超微形态似有变化,还存在其他物质的伴随分泌,故甘氨酸可能是引起细胞壁结构的改变而导致邻苯二酚2,3-双加氧酶等胞内容物被动分泌至胞外.     

固定化胞外邻苯二酚1，2－双加氧酶的研究

首次将胞外邻苯二酚1,2－双加氧酶固定化,并用于制备顺,顺—己二烯二酸.该固定化酶表观活力高,使用范围扩大,耐酸性及耐碱性都有显著提高,并且使用稳定性好,得到的产物浓度及纯度均较高,酶与产物容易分离,整个工艺简单、独特、新颖,有利于工业化应用.     

三氯卡班降解菌株Sphingomonas sp. YL-JM2C中多个邻苯二酚双加氧酶的功能

【目的】研究Sphingomonas sp.YL-JM2C菌株的生长特性,确定以三氯卡班作为碳源的生长情况。挖掘菌株YL-JM2C潜在的邻苯二酚1,2-双加氧酶及邻苯二酚2,3-双加氧酶基因,在大肠杆菌(Escherichia coli)中异源表达邻苯二酚双加氧酶基因并研究其酶学性质。【方法】优化S.sp.YL-JM2C菌株以三氯卡班作为碳源时的培养条件,并利用全自动生长曲线测定仪测定菌株生长情况,绘制生长曲线。通过生物信息学方法挖掘潜在的邻苯二酚双加氧酶基因,并分别在Escherichia coli BL21(DE3)中进行异源表达,通过AKTA快速纯化系统纯化蛋白,分别以邻苯二酚、3-和4-氯邻苯二酚为底物检测重组蛋白的酶学特性。【结果】菌株在pH为7.0-7.5时生长最优。在以浓度为4-8 mg/L的三氯卡班做为底物时,菌株适宜生长。当R2A培养基仅含有0.01%酵母提取物和无机盐时,加入终浓度为4 mg/L的三氯卡班可促进菌株生长。挖掘到6个潜在的邻苯二酚双加氧酶基因stcA1、stcA2、stcA3、stcE1、stcE2和stcE3,表达并通过粗酶液分析证明其中5个基因stcA1、stcA2、stcA3、stcE1和stcE2编码的酶均具有邻苯二酚双加氧酶和氯邻苯二酚双加氧酶的活性;纯化酶的底物范围研究揭示了StcA1、StcA2和StcA3均属于Ⅱ型邻苯二酚1,2-双加氧酶,StcE1和StcE2为两个新型邻苯二酚2,3-双加氧酶;它们酶动力学分析研究证明了5个酶对邻苯二酚的亲和力和催化效率最高,4-氯邻苯二酚次之。【结论】在同一菌株中发现了5个具有功能的邻苯二酚双加氧酶基因,stcA1、stcA2和stcA3编码的酶均属于Ⅱ型邻苯二酚1,2-双加氧酶,stcE1和stcE2为两个新型邻苯二酚2,3-双加氧酶编码基因。5个酶均具有催化邻苯二酚和氯邻苯二酚开环反应的功能,这为更好地理解微生物基因组内代谢邻苯二酚及其衍生物氯代邻苯二酚基因的多样性奠定了基础。     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.     

Cloning and expression of thermophilic catechol 1,2-dioxygenase gene (catA) from Streptomyces setonii

Streptomyces setonii (ATCC 39116) degrades various single aromatic compounds such as phenol or benzoate via an ortho-cleavage pathway using catechol 1,2-dioxygenase (C12O). A PCR using degenerate primers based on the conserved regions of known C12O-encoding genes amplified a 0.45-kbp DNA fragment from S. setonii total DNA. A Southern hybridization analysis and size-selected DNA library screening using the 0.45-kbp PCR product as a probe led to the isolation of a 6.4-kbp S. setonii DNA fragment, from which the C12O-encoding genetic locus was found to be located within a 1.4-kbp DNA fragment. A complete nucleotide sequencing analysis of the 1.4-kbp DNA fragment revealed a 0.84-kbp open reading frame, which showed a strong overall amino acid similarity to the known high-G+C Gram-positive (but significantly less to the Gram-negative) bacterial mesophilic C12Os. The heterologous expression of the cloned 1.4-kbp DNA fragment in Escherichia coli demonstrated that this C12O possessed a thermophilic activity within a broad temperature range (up to 65 degrees C) and showed a higher activity against 3-methylcatechol than catechol or 4-methylcatechol, but no activity against protocatechuate.     

Characterisation of a chromosomally encoded catechol 1,2-dioxygenase (E.C. 1.13.11.1) from Alcaligenes eutrophus CH34

Alcaligenes eutrophus CH34 used benzoate as a sole source of carbon and energy, degrading it through the 3-oxoadipate pathway. All the enzymes required for this degradation were shown to be encoded by chromosomal genes. Catechol 1,2-dioxygenase activity was induced by benzoate, catechol, 4-chlorocatechol, and muconate. The enzyme is most likely a homodimer, with an apparent molecular weight of 76,000 ± 500. According to several criteria, its properties are intermediate between those of catechol 1,2-dioxygenases (CatA) and chlorocatechol 1,2-dioxygenases (ClcA). The determined K _m for catechol is the lowest among known catechol and chlorocatechol dioxygenases. Similar K _m values were found for para-substituted catechols, although the catalytic constants were much lower. The catechol 1,2-dioxygenase from strain CH34 is unique in its property to transform tetrachlorocatechol; however, excess substrate led to a marked reversible inhibition. Some meta- and multi-substituted catechols behaved similarly. The determined K _m (or K _i) values for para- or meta-substituted catechols suggest that the presence of an electron-withdrawing substituent at one of these positions results in a higher affinity of the enzyme for the ligand. Results of studies of recognition by the enzyme of various nonmetabolised aromatic compounds are also discussed. Received: 20 November 1996 / Accepted: 11 April 1996     

Molecular cloning, genetic organization of gene cluster encoding phenol hydroxylase and catechol 2,3-dioxygenase in Alcaligenes faecalis IS-46

Alcaligenes faecalis IS-46 can utilize phenol as the sole carbon and energy source at concentration up to 1000 mg/l. In this report we created a cosmid library of this strain and the two clones specifying the whole L-46d type of phenol hydroxylase gene cluster were identified and characterized. Sequence analysis revealed that although the overall gene organization of the clusters was quite similar, few coding sequences differed or were found to have two copies compared with other source organisms. One of these coding sequences showed a good protein sequence similarity to a hypothetical protein and one matched with a regulatory protein of the LysR system. Their putative role in phenol degradation was discussed. Bioinformatic analysis suggested tentative phylogenetic assignments of the retrieved clusters. This work described for first time the complete nucleotide sequence and genetic organization of the whole phenol hydroxylase gene cluster in A. faecalis species.     

Non-invasive detection and monitoring of estrus,pregnancy and the postpartum period in pygmy loris (Nycticebus pygmaeus) using fecal estrogen metabolites

Estrone-conjugates (E₁C) were measured in the feces of six female pygmy lorises (Nycticebus pygmaeus) during estrus (n = 12), pregnancy (n = 4) and the postpartum period (n = 3). Noninvasive feces collection permitted frequent sampling throughout estrus and pregnancy, without disturbance of animals. The estrous period was defined as an increase in fecal E₁C levels above an average of 70 ng/g feces with peaks above 100 ng/g feces obtained in consecutive fecal samples collected over a 6- to 11-day period between the end of July and the first third of October. Comparison of the periovulatory profile of E₁C and the stage of labial opening of the vagina revealed a high agreement (P < 0.001). In all pregnant females, an E₁C rise was found approximately 47 days postestrus, the source of which may be the growing fetal placental unit. Estimated gestation lengths ranged between 187 and 198 days (n = 4). Am. J. Primatol. 41:103–115, 1997. © 1997 Wiley-Liss, Inc.     

Properties of cyanogen bromide-activated, Agarose-immobilized catechol 1,2-dioxygenase from freeze-dried extracts of Nocardia sp. NCIB 10503

Catechol 1,2-dioxygenase [catechol: oxygen 1,2-oxidoreductase (decyclizing); EC 1.13.11.1], the aromatic intradiol ring-cleaving enzyme of Nocardia sp. NCIB 10503 prepared by freeze-drying cell-free extracts, was covalently attached to cyanogen bromide-activated Agarose. The properties of the immobilized enzyme were compared to those of the free enzyme preparation. Immobilization was shown to increase the thermal stability of the enzyme. The pH-activity profile was altered by immobilization. Various explanations for this phenomenon are discussed. The V_max and K_m of the enzyme were not significantly affected on immobilization. The enzyme had a broader substrate specificity than any previously reported catechol 1,2-dioxygenase, and this was largely unaltered by immobilization. The properties of the preparations are compared to those of other (free) catechol 1,2-dioxygenases. The results presented show that the immobilization of catechol 1,2-dioxygenase offers an attractive means for the production of cis,cis-muconate and novel substituted analogues.