Characterization of a recombinant thermostable D-lyxose isomerase from Dictyoglomus turgidum that produces D-lyxose from D-xylulose

A putative d-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for d-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by gel-filtration chromatography. The activity of the enzyme was highest for d-lyxose, suggesting that it is a d-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co²⁺, with a half-life of 108 min, K _m of 39 mM, and k _cat of 3,570 1/min. The enzyme is the most thermostable d-lyxose isomerase among those characterized to date. It converted 500 g d-xylulose/l to 380 g d-lyxose/l after 2 h. This is the highest concentration and productivity of d-lyxose reported thus far.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Characterization of a novel thermostable l-rhamnose isomerase from Thermobacillus composti KWC4 and its application for production of d-allose

d-Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l-Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d-allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn²⁺ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (K_m), turnover number (k_cat) and catalytic efficiency (k_cat/K_m) for l-rhamnose were 33.8 mM, 1189.8 min⁻¹ and 35.2 min⁻¹ mM⁻¹, respectively. At a higher temperature, Mn²⁺ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (T_m) of T. composti L-RI was increased by 3 °C in presence of Mn²⁺. Although the T. composti L-RI displayed the optimum substrate as l-rhamnose, it could also effectively catalyze the isomerization between d-allulose and d-allose. When the reaction reached equilibrium, the sole product d-allose was produced from D-alluose by T. composti L-RI.     

Production of l-rhamnulose,a rare sugar,from l-rhamnose using commercial immobilized glucose isomerase

Abstract

A commercial immobilized d-glucose isomerase from Streptomyces murines (Sweetzyme) was used to produce l-rhamnulose from l-rhamnose in a packed-bed reactor. The optimal conditions for l-rhamnulose production from l-rhamnose were determined as pH 8.0, 60?°C, 300?g L^?1 l-rhamnose as a substrate, and 0.6?h^?1 dilution rate. The half-life of the immobilized enzyme at 60?°C was 809?h. Under the optimal conditions, the immobilized enzyme produced an average of 135?g L^?1 l-rhamnulose from 300?g L^?1 l-rhamnose after 16 days at pH 8.0, 60?°C, and 0.6?h^?1 dilution rate, with a productivity of 81?g/L/h and a conversion yield of 45% in a packed-bed reactor.     

Molecular characterization of a thermostable L-fucose isomerase from Dictyoglomus turgidum that isomerizes L-fucose and D-arabinose

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn²⁺ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k_cat of 15,500 min⁻¹ and a K_m of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k_cat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K_m, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k_cat, but had little effect on K_m.     

Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum

Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40 kDa) and CD structural studies showed a conserved α/β structure at different temperatures (25 and 70 °C) and high thermoresistance (Tm of 88 °C). Interestingly, the enzyme showed high endo-β-1,4-mannanase activity vs various mannans, but low endo-β-1,4 glucanase activity towards carboxymethylcellulose. The K _M and V _max of DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 μmol/min mg and 1.83 mg/ml and 1.349 μmol/min mg, respectively. Its optimal activity towards temperature and pH resulted to be 70 °C and pH 5.4, respectively. Further characterization highlighted good thermal stability (~ 50% of enzymatic activity after 2 h at 70 °C) and pH stability over a broad range (> 90% of activity after 1 h in buffer, ranging pH 5–9); resistance to chemicals was also observed.

     

Characterization of Mesorhizobium loti L-rhamnose isomerase and its application to L-talose production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

d-lyxose isomerase and its application for functional sugar production

Applied Microbiology and Biotechnology - Functional sugars have attracted attention because of their wide application prospects in the food, cosmetics, and pharmaceutical industries in recent...     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.     

Production of d-allose from d-fructose using immobilized l-rhamnose isomerase and d-psicose 3-epimerase

Bioprocess and Biosystems Engineering - d-Allose is a rare sugar, can be used as an ingredient in a range of foods and dietary supplements, has alimentary activities, especially excellent...     

链霉菌来源D-甘露糖异构酶的性质及其在制备D-甘露糖中的应用

[背景]D-甘露糖具有多种功能活性,在食品、医药、饲料等行业应用广泛.D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的酶法制备中具有应用潜力.[目的]克隆一个链霉菌(Streptomyces sp.)来源的D-甘露糖异构酶基因(sssMIaseA)并在大肠杆菌中表达,研究其酶学性质,并用于制备...     

发酵乳杆菌来源l-阿拉伯糖异构酶理性设计及在d-塔格糖生产中的应用

L-阿拉伯糖异构酶(L-arabinose isomerase,L-AI)是D-半乳糖异构化生成D-塔格糖的关键酶。为提高L-阿拉伯糖异构酶对D-半乳糖的活性及在生物转化中的转化率,本研究对发酵乳杆菌(Lactobacillus fermentum)CGMCC2921来源的L-阿拉伯糖异构酶进行重组表达和生物转化应用,并对其底物结合口袋进行理性设计以提高酶对D-半乳糖亲和力和催化活性。结果显示,突变体F279I对D-半乳糖的转化率提高至野生型酶的1.4倍,进一步叠加获得的双突变体M185A/F279I的K_m和k_cat分别为530.8mmol/L与19.9s^-1,底物亲和力显著提高,催化效率提高至野生型酶的8.2倍。以400 g/L乳糖为底物时,突变酶M185A/F279I转化率高达22.8%。本研究在乳糖高值化生产塔格糖方面具有重要的应用价值。     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.     

重组大肠杆菌单链结合蛋白性质表征及其在提高焦磷酸测序准确性中的应用

利用基因工程手段表达了分子量约为24 kDa的重组大肠杆菌单链结合蛋白 (r-SSBP),通过凝胶阻滞电泳与DNA熔解温度 (Tm) 影响实验表征了r-SSBP与单链DNA (ssDNA) 结合的特性,结果表明,r-SSBP可以与ssDNA结合,并且能够降低DNA的Tm值,同时还能增大含有单个错配碱基的DNA与完全匹配的DNA的Tm值差异,这一特性在提高单核苷酸多态性检测的特异性方面具有潜在的应用价值。此外,将r-SSBP应用于本课题组开发的高灵敏度焦磷酸测序体系中测定已知序列ssDNA模板,结果表明,r     

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.     

L-Arabinose isomerase and its use for biotechnological production of rare sugars

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.     

苏云金芽孢杆菌重组L-异亮氨酸羟化酶的酶学性质及其在4-羟基异亮氨酸合成中的应用

【目的】克隆并表达来源于苏云金芽孢杆菌(Bacillus thuringiensis)TCCC 11826的L-异亮氨酸羟化酶(L-isoleucine-4-hydroxylase,IDO),测定重组IDO酶学特性并构建用于4-羟基异亮氨酸(4-Hydroxyisoleucine,4-HIL)微生物转化的重组菌株,以考察该酶在4-HIL合成中的潜在应用价值。【方法】以B.thuringiensis TCCC 11826基因组为模板PCR扩增ido基因并构建该基因过表达菌株BL-IDO;采用Ni-NTA亲和层析法分离纯化重组IDO后检测其酶学特性;构建重组株菌W3110-IDO进行4-HIL的微生物转化。【结果】克隆B.thuringiensis TCCC 11826的ido基因,测序结果显示该基因含723个核苷酸,编码240个氨基酸,与已报道的B.thuringiensis 2-e-2的ido基因相似度分别为97.47%和97.91%。此IDO含有His1-X-Asp/Glu-Xn-His2基序,属于Fe2+和α-酮戊二酸依赖型羟化酶家族;酶学实验表明该酶能够特异性地催化L-异亮氨酸生成(2S,3R,4S)-4-HIL,其Km和Vm ax分别为0.18 mmol/L和2.10μmol/min/mg,最适反应温度和pH分别为35℃和7.0,该酶于35℃条件下放置5 h后仍具有85.1%的活性;在Escherichia coli W3110中过表达重组IDO,在未经优化条件下4-HIL最高转化率达89.28%。【结论】获得IDO编码基因序列(Accession No.KC884243)并首次较为系统地研究了其酶学特性,该酶反应条件温和且具有较高的活性及稳定性,在酶法或微生物转化法合成4-HIL中有较广泛的应用价值。本研究可为4-HIL及其它氨基酸衍生物的生物制造技术奠定理论基础。