厌氧菌Sunxiuqinia sp. SH-52外切型海藻酸裂解酶的异源表达及酶学特征

【背景】目前已报道的海藻酸分解菌多数为好氧菌,未见有关厌氧菌的报道。从分离的海藻酸分解菌中表征的海藻酸裂解酶大多为内切型海藻酸裂解酶,外切型较少。【目的】研究来自厌氧海藻酸分解菌的海藻酸裂解酶基因,表征新型海藻酸裂解酶并阐明其酶学性质,为海藻酸裂解酶的多样性和微生物降解海藻酸机制提供理论依据。【方法】对来自厌氧海藻酸分解菌Sunxiuqinia sp. SH-52的海藻酸裂解酶SHA-4编码基因进行克隆,分析基因序列,构建重组质粒PGEX-4T-1-SHA-4并在大肠杆菌中实现异源表达,经纯化后对其酶学性质及降解特征进行研究。【结果】该酶在28°C、用0.1 mmol/L IPTG (异丙基-β-D-硫代半乳糖苷)条件下诱导6 h达到最大表达量,纯化后酶的比活力达到21 U/mg。酶学性质分析表明SHA-4的最适温度为37°C;最适pH为7.5;对PolyMG (杂聚古罗糖醛酸-甘露糖醛酸嵌段)具有底物偏好性;Na+对该酶的活性具有抑制作用,而金属离子Cu~(2+)具有明显促进作用,使活性提高了约168%;SHA-4催化海藻酸的Km值为2.5 mg/mL,Vmax为8.7 mg/(mL·min);SHA-4为外切型海藻酸裂解酶,降解海藻酸终产物为单糖。【结论】异源表达了来自一株厌氧海藻酸分解菌Sunxiuqiniasp.SH-52的海藻酸裂解酶SHA-4,该酶是PL6家族中第一个对PolyMG有底物偏好性的外切型海藻酸裂解酶,而且活性较高,作为工具酶有很好的应用前景,为海藻酸降解机制的探索提供了新的线索。     

Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.

The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.     

Cloning of alginate lyase gene (alxM) and expression in Escherichia coli.

The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.     

Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli

An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg²⁺. The enzyme was poly -D-1, 4-mannuronate-specific rather than -D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly -D-1, 4-mannuronate-specific alginate lyase from the same strain.     

Cloning of alginate lyase gene (alxM) and expression in Escherichia coli

The alxM gene encoding a D-mannuronan-specific alginate lyase has been cloned from a marine bacterium isolated as an epiphyte on the brown alga, Sargassum fluitans. Expression of this gene in Escherichia coli provides a source of this enzyme for probing alginate structure and modifying the mannuronan-rich alginate polymers produced by bacterial pathogens.     

Cloning and Characterization of Alginate Lyase from a Marine Bacterium Streptomyces sp. ALG-5

A marine bacterium was isolated from seaweeds for its ability to degrade alginate. Analysis of 16S ribosomal DNA sequence and chemotaxonomic characterizations revealed that the strain belongs to Streptomyces. The alginate lyase gene of Streptomyces sp. ALG-5 was cloned by using PCR with the specific primer designed from homologous nucleotide sequences. The consensus sequences of N-terminal YXRSELREM and C-terminal YFKAGXYXQ were conserved in the ALG-5 alginate lyase gene. The recombinant alginate lyase was purified by using Ni-Sepharose affinity chromatography. The alginate lyase appears to be poly-guluronate lyase degrading poly-G block preferentially than poly-M block. The degraded products were determined to be di-, tri-, tetra- and pentasaccharides by using BioGel P-2 gel filtration chromatography and ionization mass spectroscopy method.     

Purification and characterization of the recombinant alginate lyase from Pseudomonas sp. leaked by Escherichia coli upon addition of glycine

Abstract The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp. OS-ALG-9, fused in frame to the β-galactosidase α-peptide gene. The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine. The extracellular enzyme isolated from the culture of E. coli JM109 (pAL205) was purified over 15 000-fold by successive chromatography and subjected to amino acid sequence analysis. The sequence determined was identical to that of the intracellular protein. Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas , the glycine does not affect or modify the Aly during its leakage into the medium.     

内生菌Pseudomonas sp. G5 phzIR基因的克隆与表达

假单胞菌菌株G5是分离自香菜(Coriandrum sativumL.)茎内的一株内生菌,经BIOLOG系统分析其底物利用图谱,初步鉴定为桔黄假单胞菌Pseudomonas aurantiaca。大量研究已表明许多革兰氏阴性细菌应用群体感应系统,通过感应扩散性小信号分子―乙酰基高丝氨酸内酯(N-acyl homoserine lactones,AHLs),以种群密度依赖的方式调控基因表达,控制植物相关细菌的多种表型。本研究组合应用AHLs检测菌株Chromobacterium violaceum CV026和薄层层析分析,初步检测出菌株G5可产生几种可检测水平的AHLs信号分子,其中以N-hexanoyl-homoserine lactone(C6-HSL,HHL)为主,迁移率Rf值为0.4。进一步克隆和测序了该菌株中由PhzI和PhzR组成的群体感应quorumsensing系统的编码基因phzIR,并在大肠杆菌中异源表达了AHLs信号分子合成酶基因phzI。序列和系统进化分析表明它们与假单胞菌属其他的phzIR基因有高度同源性和进化上的保守性。     

Control and heterologous expression in Escherichia coli of the Klebsiella pneumoniae gene encoding alginate lyase

The gene (aly) encoding the guluronate-specific alginate lyase from Klebsiella pneumoniae has been subcloned into the plasmid vector pHG327, transformed into Escherichia coli, and expressed in this host. Three groups of lyase-positive clones have been identified in which one or more copies of a 1.95-kb Hind III fragment encoding the gene have been inserted. The enzyme is expressed constitutively from its own promoter, but the efficiency depends on the orientation of the insert within the vector. Similarly, enhanced expression by induction of the vector's lac promoter is also orientation-dependent.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli.

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.     

Heterologous expression of murine lymphotoxins in Escherichia coli and preparation of antibodies]

Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E. coli cells. The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT.     

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.     

An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli,purification, and characterization of alginate lyase IV (A1-IV)

Sphingomonas sp. A1 (strain A1) cells contain three kinds of endotype alginate lyases [A1-I, A1-II, and A1-III], all of which are formed from a common precursor through posttranslational processing. In addition to these lyases, another type of lyase (A1-IV) that acts on oligoalginates exists in the bacterium. A1-IV was overexpressed in Escherichia coli cells through control of its gene under the T7 promoter. The expression level of the enzyme in E. coli cells was 8.6U/L-culture, which was about 270-fold higher than that in strain A1 cells. The enzyme was purified to homogeneity through three steps with an activity yield of 10.9%. The optimal pH and temperature, thermal stability, and mode of action of the purified enzyme were similar to those of the native enzyme from strain A1 cells. A1-IV exolytically degraded oligoalginates, which were produced from alginate through the reaction of A1-I, A1-II, or A1-III, into monosaccharides, indicating that the cooperative actions of these four enzymes cause the complete depolymerization of alginate in strain A1 cells.     

褐藻胶裂解酶基因的克隆表达与酶学性质

随着大型褐藻生产燃料乙醇以及褐藻寡糖重大药用价值的发现,褐藻胶裂解酶成为国内外多个领域的研究重点。文中对解藻酸弧菌上与褐藻胶降解相关的5个基因分别进行克隆表达,通过SDS-PAGE和酶活性定量测定,发现该基因簇中的4个基因有降解褐藻胶活性。对酶活最高的rAlgV3进行了诱导条件的优化、酶蛋白纯化及酶性质研究,发现优化诱导条件后重组酶rAlgV3的酶活由2.34×10~4 U/L上升为1.68×10~5 U/L,比优化前提高了7.3倍;对酶性质进行表征发现该酶在4–70℃均有活性,最适反应温度为40℃,在4–20℃酶相对稳定;该酶在pH 6.5-9.0环境下均有较高的酶活,最适pH为8.0;pH稳定性好,在pH 4.5–9.5环境下可以稳定存在;适量的NaCl浓度和Fe~(2+)、Fe~(3+)等离子具有促进酶活的作用,SDS和Cu~(2+)离子可明显抑制酶活力。对该酶的底物特性的研究发现,该酶不仅可以降解褐藻胶中的Poly-M片段,也能降解Poly-G片段,具有广泛底物特性;其降解海藻酸钠主要释放二糖和三糖,是一种内切酶。该酶对于第三代燃料乙醇的发展及褐藻寡糖的生产具有重要作用。     

天蓝色链霉菌海藻糖合酶的异源表达、活性分析及重组菌全细胞转化合成海藻糖的条件优化

从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因 (ScTreS),在大肠杆菌Escherichia coli BL21(DE3) 中进行了异源表达,通过 Ni-NTA 亲和柱对表达产物进行分离纯化得到纯酶,经 SDS-PAGE 测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35 ℃;最适pH 7.0,对酸性条件比较敏感。通过同源建模和序列比对分析,对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍,突变酶A165T相对提高了1.39倍,海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5 L罐发酵,结果表明：在麦芽糖浓度300 g/L、初始反应温度和pH分别为35 ℃和7.0的条件下,转化率最高达到71.3%,产量为213.93 g/L;当底物浓度增加到700 g/L时,海藻糖产量仍可达到465.98 g/L。     

A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65,000. The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69,543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.     

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli

Abstract A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the d-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M _r 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli .