Enzymatic Properties of Recombinant Kojibiose Phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP≧4 than KP from Thermoanaerobacter brockii ATCC35047.     

Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-d-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 °C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 °C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70 °C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.     

Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus

Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed.     

Construction and characterization of chimeric enzymes of kojibiose phosphorylase and trehalose phosphorylase from Thermoanaerobacter brockii

Chimeric phosphorylases were constructed of the kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.     

Hydrolytic properties of a thermostable α‐l‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg^?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum k_cat/K_m of 220 m(mol l^?1)^?1 s^?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l^?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml^?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase.     

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.     

纤维二糖差向异构酶在毕赤酵母中的表达及酶学性质研究

首先将来源于Caldicellulosiruptor saccharolyticus的纤维二糖差向异构酶基因CsCEm进行密码子优化,然后进行全基因合成,再将其引入到载体pPIC9K中,构建重组质粒pPIC9K-CsCEm并转化入毕赤酵母GS115,得到酵母工程菌株.经微孔板筛选、摇瓶筛选得到酶活最高的重组工程茵GS115-4-19.该菌株经甲醇诱导144 h后,摇瓶发酵液上清酶活达到0.42 U/mL.酶学性质研究结果表明:该酶的最适pH为7.5,且在pH 6.0 ～8.0范围内相对酶活都在80％以上;在pH 4～9的缓冲液中放置24 h后仍保持原酶活力的80％以上;最适温度为80℃,在60℃～80℃保温30 min后,相对酶活在80％以上.动力学研究结果表明该酶对底物乳糖的Km和Vmax分别为(120.27±9.96) mmol/L和(1.035±0.05) mmol/L/min.纤维二糖差向异构酶在毕赤酵母中的成功表达为生物酶法合成乳果糖提供了重要参考.     

Uridine phosphorylase from Escherichia coli B. Enzymatic and molecular properties

Uridine phosphorylase (EC 2.4.2.3) from Escherichia coli B is an oligomeric protein composed of four identical subunits of 29,000 mol. wt. The enzyme has four half-cystine residues per subunit titrable only in denaturing condition. No disulphide linkages either inter- or intra-chain are present. The isoelectric point is 5.25. The enzyme shows strict specificity toward uridine and 5-methyluridine and is inhibited by thymine, deoxycytidine and heavy metal ions.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus

A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides d-fucose, d-xylose, l-arabinose, and d-galactose, as well as on l-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor d-glucose, resulting in drastically increased K(m) values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.     

Cloning and sequence of a type I pullulanase from an extremely thermophilic anaerobic bacterium,Caldicellulosiruptor saccharolyticus

A gene coding for a pullulanase from the obligately anaerobic, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus has been cloned in Escherichia coli. It consists of an open reading frame (pulA) of 2478 bp which codes for an enzyme of 95 732 Da and is flanked by two other open reading frames. A truncated version of the gene which lacks 381 bp of 5′-sequence also has pullulanase activity and it appears that the amino-terminal portion of the gene is not essential for either activity or thermostability. Amino acid sequence comparisons with other published amylases and pullulanases showed that it possesses homology to the four key regions common to these enzymes.     

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBP_Bl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k _cat/K _m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBP_Bl showed similar k _cat/K _m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.     

解糖热解纤维素菌F32降解未预处理秸秆及产纤维素酶特性分析

【目的】明确极端嗜热厌氧木质纤维素降解菌解糖热解纤维素菌F32代谢特征,并分析其产酶特性。【方法】使用细胞计数法绘制菌株的生长曲线,使用离子色谱及气相色谱进行产物和残糖量分析,以DNS法及对硝基苯酚法检测菌株胞外蛋白的酶活性。【结果】解糖热解纤维素菌F32在以葡萄糖、微晶纤维素和未经预处理小麦秸秆为碳源时生长状况优于解糖热解纤维素菌DSM 8903。在以葡萄糖为碳源进行培养时,与菌株DSM 8903相比,菌株F32具有产乳酸较多,而产氢气较少的特点。在以微晶纤维素和未经预处理小麦秸秆为碳源进行培养时,与菌株DSM 8903相比,菌株F32胞外蛋白具有较高的内切纤维素酶活性和木聚糖酶活性。【结论】解糖热解纤维素菌F32表现出较强的木质纤维素降解能力,其与DSM 8903的产物组成及胞外蛋白的酶活性具有明显差异。     

蔗糖富集环境中蔗糖磷酸化酶的酶学性质研究与改造

【背景】蔗糖富集土壤是蔗糖磷酸化酶的重要来源之一。此酶能以较廉价的蔗糖为底物进行转葡萄糖基反应,改善受体底物的理化性质,因而具有重要的应用价值。【目的】研究蔗糖富集土壤中蔗糖磷酸化酶的酶学性质和转糖苷活性,为其更优质的改造提供材料和理论基础。【方法】将宏基因组中获得的蔗糖磷酸化酶基因克隆到表达载体上构建重组大肠杆菌,诱导表达并进行镍亲和层析纯化蛋白。以蔗糖为底物测量重组酶的基本酶学性质,研究其对糖类底物的转糖苷活性。通过底物通道分析,利用反向PCR技术对其第155位点进行饱和突变,并测定突变体基本酶学性质和转糖苷活性。【结果】纯化的酶蛋白分子量大小约为56 kD,活性状态时以三聚体形式存在。在以蔗糖为底物时,最适温度和最适pH值分别为55°C和6.5;Km和Vmax值分别为23.1±2.4 mmol/L和407.9±8.5μmol/(mg·min),对第155位点进行了定点饱和突变,获得了部分酶学性质或转糖苷活性改善的突变体。【结论】宏基因组中蔗糖磷酸化酶的研究丰富了酶学数据,并通过分子改造获得了部分性质更优的突变体,为转糖苷活性关键氨基酸的研究和该酶的工业应用奠定了基础。     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2′-脱氧核苷

6-甲基嘌呤-2′-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞。本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2′-脱氧尿苷为底物合成6-甲基嘌呤-2′-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%。用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%。HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR。     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2'-脱氧核苷

6-甲基嘌呤-2'-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞.本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2'-脱氧尿苷为底物合成6-甲基嘌呤-2'-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%.用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%.HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR.     

Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

Background  

The production of hydrogen from biomass by fermentation is one of the routes that can contribute to a future sustainable hydrogen economy. Lignocellulosic biomass is an attractive feedstock because of its abundance, low production costs and high polysaccharide content.