Heterologous expression, purification, and characterization of a highly active xylose reductase from Neurospora crassa

A xylose reductase (XR) gene was identified from the Neurospora crassa whole-genome sequence, expressed heterologously in Escherichia coli, and purified as a His6-tagged fusion in high yield. This enzyme is one of the most active XRs thus far characterized and may be used for the in vitro production of xylitol.     

Purification,characterization and thermostability of lipase from a thermophilic Bacillus sp. J33

A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The K_m and V_max for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 M min^-1 ml^-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.     

蛇足石杉内生真菌Shiraia sp. Slf14中赖氨酸脱羧酶基因的克隆、表达及功能

【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。     

Pyrone derivatives from the marine-derived fungus Nigrospora sp. PSU-F18

Pyrones, named nigrosporapyrones A-D (1-4), and five known compounds were isolated from the marine-derived fungus Nigrospora PSU-F18. Their structures were elucidated on the basis of spectroscopic evidence. The antibacterial activity against the standard Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus was evaluated.     

Purification and characterization of thermo-labile alkaline phosphatase from an Antarctic psychrotolerant Bacillus sp. P9

A psychrotolerant Bacillus sp. from Antarctica produced an alkaline phosphatase in the culture supernatant. The strain showed 98.4% 16s rDNA sequence identity with Bacillus sphaericus. The 76 kDa protein was purified 11.1-fold showing alkaline phosphomonoesterase activity. Enzyme was optimally produced at 25 °C and pH 7.0. This cold active alkaline phosphatase is heat labile and gets completely inactivated at 60 °C in 50 min and is active in broad pH range.     

热稳定酯酰辅酶A合成酶的异源表达及酶学特性

【目的】为寻找能合成丙酰辅酶A和丁酰辅酶A等聚酮合成前体的生物催化剂,用体外酶学实验对一个酯酰辅酶A合成酶进行了表征。【方法】利用丙二酰辅酶A合成酶作为输入序列,通过BLAST程序在Caldicellulosiruptor owensensis OL的基因组中找到1个酯酰辅酶A合成酶基因。在大肠杆菌中进行了异源表达,并通过亲和层析进行纯化。底物谱、最适反应条件、稳定性和动力学参数通过体外酶学实验进行表征,而定点突变则用于活性中心的氨基酸残基的分析。【结果】该酶具有较好的底物宽泛性,可识别丙酸、丁酸、2-甲基丙酸、戊酸、3-甲基丁酸、2-甲基丁酸以及环己甲酸等一系列单酸。反应最适温度为30°C,最适p H为7.0。70°C保温8 h后仍有45%的活性残留,表明该酶相对比较稳定。通过活性中心3个位点的定点突变可以改变酶的底物特异性。【结论】C.owensensis OL来源的酯酰辅酶A合成酶是潜在的生物催化剂,可以用于聚酮前体的合成。     

Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys–Gly–Pro–Cys active-site sequence. After purification by a single step Ni–NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0–10 °C. It had high tolerance to a wide range of NaCl concentrations (0–2 M NaCl) and was stable in the presence of H₂O₂. This research suggested that PsTrx displayed unique catalytic properties.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C₂ to C₁₂ and the preferred substrate was pNP-pentanoate showing a k_cat = 26.2 ± 0.1 s⁻¹, K_M = 0.122 ± 0.006 mM and a k_cat/K_M = 215 ± 11 mM⁻¹ s⁻¹. The enzyme was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Mn²⁺ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C₅₀ values of 1.98 M and 0.92 M, respectively.     

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.     

一株红树林曲霉菌Aspergillus sp.WHUF0343的次级代谢产物研究

【目的】研究分离自三亚亚龙湾红树林根系土壤的真菌Aspergillus sp. WHUF0343的次级代谢产物及其生物活性。【方法】利用硅胶柱层析、Sephadex LH-20凝胶柱层析和半制备高效液相色谱等技术对该菌的发酵产物进行分离纯化;综合利用核磁共振波谱(NMR)和质谱(MS)等现代波谱技术以及与文献数据对照确定化合物的结构;分别采用肉汤微量稀释检测法和MTS法对化合物进行抗菌和肿瘤细胞毒活性测试。【结果】从真菌Aspergillus sp.WHUF0343的发酵产物共分离并鉴定了10个化合物,分别为isoechinulin A (1)、neoechinulin A (2)、neoechinulin E (3)、preechinulin (4)、neoechinulin D (5)、variecolorin J (6)、dehydroechinulin (7)、questinol (8)、emodin (9)和catenarin (10)。活性评价显示化合物2、9和10对幽门螺杆菌(Helicobacter pylori)和金黄色葡萄球菌(Staphylococcus aure...     

Abditibacterium utsteinense sp. nov., the first cultivated member of candidate phylum FBP,isolated from ice-free Antarctic soil samples

Most bacterial lineages are known only by molecular sequence data from environmental surveys and represent the uncultivated majority. One of these lineages, candidate phylum FBP, is widespread in extreme environments on Earth, ranging from polar and desert ecosystems to wastewater and contaminated mine sites. Here we report on the characterization of strain LMG 29911^T, the first cultivated representative of the FBP lineage. The strain was isolated from a terrestrial surface sample from Utsteinen, Sør Rondane Mountains, East Antarctica and is a Gram-negative, aerobic, oligotrophic chemoheterotrophic bacterium. It displays growth in a very narrow pH range, use of only a limited number of carbon sources, but also a metabolism optimized for survival in low-nutrient habitats. Remarkably, phenotypic and genome analysis indicated an extreme resistance against antibiotics and toxic compounds. We propose the names Abditibacterium utsteinense for this bacterium and Abditibacteriota for the former candidate phylum FBP. Furthermore, inter- and intra-phylum relationships indicate Armatimonadetes, a neighboring lineage to the Abditibacteriota, to be a superphylum.     

Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes

Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40–60 g l⁻¹ ethanol from 15 % (w/v) d-glucose, d-fructose, d-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83 % observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15 % (w/v) SCBC with commercial cellulase was approximately 20 %. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15 % SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g⁻¹ biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6 %. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.     

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81 kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35 °C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35 °C for 1 h. Higher activity was observed in the presence of surfactants, Na⁺, NH₄⁺ ions, NaN₃ and ethylenediaminetetraacetic acid (EDTA), while Co²⁺ and Cu²⁺ ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10 days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.     

芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质

对一株从土壤中分离到的芽胞杆菌Bacillus sp.BSD-8菌株所产生的热稳定性较高的肌氨酸氧化酶进行纯化,并对该酶的特性进行了研究。通过硫酸铵分级沉淀、DEAE-纤维素离子交换柱、Toyopearl疏水层析柱和Sephadex G-75分子筛层析,使酶提纯25倍,比活力达到5.3U/mg。研究了纯化后的酶的生化特性,确定了该酶的主要特性:该酶为黄素蛋白,与黄素以非共价键的方式结合,由单一亚基组成,其亚基分子量为51kDa。酶的最适反应温度及pH分别为60℃与8.5。该酶在60℃及pH8.0~10.0条件下稳定。以Lineveaver-Burk作图法求得该酶米氏常数Km值为3.1mmol/L。Ag+、Hg2+、SDS及Tween80对该酶有强抑制作用,而Tween20和Triton X-100对酶活性无影响。该肌氨酸氧化酶在耐热性质上比以前所报道的肌氨酸氧化酶有很大的提高,在酶法肌酐测定应用中有明显的优势。     

菊粉酶基因的异源表达、分离纯化及酶学性质

为进一步提高菊粉酶在生物技术领域的应用,研究了来源于马克斯克鲁维酵母Kluyveromyces marxianus YX01的菊粉酶性质。通过在毕赤酵母GS115宿主细胞中异源表达该菊粉酶基因(inu),获得了一种外切型菊粉酶,经聚丙烯酰胺凝胶电泳(SDS-PAGE)验证其分子量为86.0 k Da。进一步在该菊粉酶上增加6个His标签,采用聚乙二醇(PEG)20 000透析浓缩和Ni-NTA Agarose静态亲和吸附作用的方法,完成菊粉酶的分离纯化,纯化倍数和酶回收率分别为3.6和33.1%。比较发现粗酶液与纯酶的酶学性质相似,且菊粉酶的最适反应温度为60℃,最适p H值为4.62,并测得该酶的Km和Vmax值,以菊粉为底物时,Km和Vmax值分别为80.53 g/L和4.49 g/(L·min);以蔗糖底物时,Km和Vmax值分别为183.10 g/L和20.20 g/(L·min)。金属离子Mn2+、Ca2+、Cu2+、Zn2+和Fe2+对酶活力具有不同程度的抑制作用,其中Cu2+、Zn2+和Fe2+的抑制作用最为显著。这些研究为进一步提高菊粉酶在工业化的应用奠定了基础。     

Survival and redox activity of Friedmanniomyces endolithicus, an Antarctic endemic black meristematic fungus,after gamma rays exposure

Despite living organisms are not exposed to acute ionizing radiation under natural conditions, some exhibit a high radiation resistance. Understanding this phenomenon is important for assessing the impact of radiation-related accidents, occupational exposures and space missions. In this context, in this study we analyzed the effect of gamma rays on the Antarctic cryptoendolithic melanized fungus Friedmanniomyces endolithicus CCFEE 5208 and demonstrated its resistance to acute doses of gamma radiation (up to 400 Gy), accompanied by increase in metabolic activity.     

A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability

Aims: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high‐yield production of enzymatic hydrogen. Methods and Results: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64·9 kDa. This enzyme was found to be a dual‐specificity enzyme – PGM/phosphomannomutase (PMM). Mg²⁺ and Mn²⁺ were activators. Ser144 was identified as an essential catalytic residue through site‐directed mutagenesis. The k_cat and K_m of PGM were 190 s^?1 and 0·41 mmol l^?1 on glucose‐1‐phosphate and 59 s^?1 and 0·44 mmol l^?1 on mannose‐1‐phosphate, respectively, at 60°C. Thermostability of PGM at a low concentration (2 nmol l^?1, 100 U l^?1) was enhanced by 12‐fold (i.e. t_1/2 = 72 h) at 60°C with addition of bovine serum albumin, Triton X‐100, Mg²⁺and Mn²⁺. Conclusions: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. Significance and Impact of the Study: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).