Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

利用固定化重组大肠杆菌细胞生产D-塔格糖

利用经海藻酸钙包埋的重组大肠杆菌细胞催化D-半乳糖生产D-塔格糖,考察了细胞包埋量、反应条件对固定化细胞催化效率以及对D-塔格糖生产稳定性的影响。确定的最优转化条件为:温度65℃,pH 6.5,添加终浓度为1 mmol/L Mn²⁺,底物(D-半乳糖)浓度100 g/L,重组大肠杆菌细胞用量40 g/L。固定化小球在0.3%戊二醛溶液中交联30 min可以显著提高其在高温下的机械强度。考察了异构化反应体系中硼酸与底物间的摩尔比对产率的影响。研究结果表明,添加适量的硼酸可以改变原有的化学反应平衡,实现D-塔格糖的高产。利用D-半乳糖为底物在最优的反应条件下催化24 h,固定化细胞对D-半乳糖的转化率最高,可达65.8%,连续转化8批次的平均转化率为60.6%,为工业化生产D-塔格糖奠定了基础。     

High production of D-tagatose by the addition of boric acid

An L-arabinose isomerase mutant enzyme from Geobacillus thermodenitrificans was used to catalyze the isomerization of D-galactose to D-tagatose with boric acid. Maximum production of D-tagatose occurred at pH 8.5-9.0, 60 degrees C, and 0.4 molar ratio of boric acid to D-galactose, and the production increased with increasing enzyme concentration. Under the optimum conditions, the enzyme (10.8 units/mL) converted 300 g/L D-galactose to 230 g/L D-tagatose for 20 h with a yield of 77% (w/w); the production and conversion yield with boric acid were 1.5-fold and 24% higher than without boric acid, respectively. In 24 h, the enzyme produced 370 g/L D-tagatose from 500 g/L D-galactose with boric acid, corresponding to a conversion yield of 74% (w/w) and a production rate of 15.4 g/L.h. The production and yield of D-tagatose obtained in this study are unprecedented.     

生物转化生产D-塔格糖的食品级菌种选育及L-阿拉伯糖异构酶的克隆表达

L-阿拉伯糖异构酶(L-arabinose isomerase,L-AI)是一种可以催化D-半乳糖为D-塔格糖的胞内异构化酶。随着塔格糖在食品工业中越来越广泛的应用,能够将半乳糖转化为塔格糖的食品级微生物以及食品级来源的L-AI受到更大的关注。文中从各种酸奶制品、泡菜及其他一些食品中采集不同的样品,筛选出1株具有L-AI酶活的食品级菌株,经过生理生化鉴定以及16S rDNA序列测定,确定该菌株为戊糖片球菌,命名为Pediococcus pentosaceus PC-5。以该菌基因组为模板,克隆L-AI基因,并在大肠杆菌BL21成功地异源表达。表达产物经粗提取后,在40℃下加入Mn2+,使D-半乳糖转化为D-塔格糖的转化率为33%。     

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.     

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K _m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe²⁺ and Mn²⁺ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu²⁺, Zn²⁺, Hg²⁺, and Fe³⁺ ions inhibited the reaction completely. In the presence of 1 mM Fe²⁺ ions, the K _m for galactose was found to be 300 mM.     

微生物来源的L-阿拉伯糖异构酶的研究进展及应用前景

L-阿拉伯糖异构酶(L-AI)能分别催化L-阿拉伯糖和D-半乳糖异构为L-核酮糖和D-塔格糖,它是目前生物法生产新型功能性因子D-塔格糖最为有效的酶.近年来,L-AI的结构已被揭晓,其基因已获得克隆、测序和过量表达,经过蛋白质工程改造的L-AI将是未来工业化生产D-塔格糖的主要用酶.本文综述了近年来国外对L-AI的结构与功能、催化机理、酶学性质及应用于D-塔格糖生产方面的研究状况,并展望了其发展前景.     

A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting ß-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from

ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.     

Bacillus Stearothermophilus IAM 11001 L-阿拉伯糖异构酶在大肠杆菌中的表达、纯化及活性研究

L-阿拉伯糖异构酶是生物法生产新型功能性因子D-塔格糖最为有效的酶。本文获得了一种新型耐热L-阿拉伯糖异构酶的编码基因araA,来源于Bacillus stearothermophilis IAM 11001,经NCBI Blastn分析,与GenBank中Thermus sp. IM6501 araA序列的同源性为95%,并将该新基因提交到GenBank,获得登陆号：EU394214。以pET-22b(+)为载体质粒,E. coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白的过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE电泳分析,约在59 kDa处出现显著的特征蛋白条带;同时对重组L-AI的活性进行了初步研究,全细胞反应24小时D-塔格糖的转化率为39.8%。     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process.     

An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization

The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 75°C and pH 7.5. It required divalent metal ions, either Mn²⁺ or Co²⁺, for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent K _m, V _max, and catalytic efficiency (k _cat/K _m) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM⁻¹ min⁻¹, respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after 12 h under optimal conditions, suggesting its potential in D-tagatose production.     

生物法生产D-塔格糖的研究进展

D-塔格糖具有多种独特的生理特性与功能,近年来已被发达国家开发作为具有高经济附加值的功能性甜味剂进行销售。D-塔格糖的商业化生产长期以来依赖化学催化法,随着20世纪90年代利用L-阿拉伯糖异构酶(简称L-AI酶)催化D-半乳糖制备D-塔格糖技术的兴起,生物法生产D-塔格糖成为了新的发展趋势。结合笔者所在课题组近年来的研究成果,就D-塔格糖生物法生产工艺的研究现状和前景进行综述与展望。     

Improved substrate specificity and dynamic range for glucose measurement of Escherichia coli PQQ glucose dehydrogenase by site directed mutagenesis

Site directed mutagenesis study was carried out with Escherichia coli pyrroloquinoline quinone glucose dehydroge-nase (PQQGDH) by substitution of His775 with either Asn (H775N) or Asp (H775D). The mutated PQQGDHs had different substrate specificity and catalytic activity from the wild type PQQGDH. The K values of H775N for 2-deoxy-D-glucose and for D-allose increased for 10-fold. The K values for both D-mannose and D-galactose were estimated much higher than 100 mM. H775D also showed the increase in K values toward saccharides. As a result, these mutants possessed narrower substrate specificity than wild type E. coli PQQGDH. H775D showed the increase in K value for glucose versus wild type PQQGDH (25-fold), therefore H775D is suitable for the direct measurement of blood glucose. The role of His775 in E. coli. PQQGDH is also discussed.     

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.     

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.     

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.     

Novel substrates of a ribose-5-phosphate isomerase from Clostridium thermocellum

A substrate specificity study of the recombinant D-ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum was performed. Among all aldopentoses and aldohexoses, the RpiB enzyme displayed activity with L-talose, D-ribose, D-allose, L-allose, L-ribose, and D-talose in decreasing order. The products released were L-tagatose, D-ribulose, D-psicose, L-psicose, L-ribulose, and D-tagatose, respectively. The enzyme showed specificity for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2, C3, and C4 positions. Molecular modeling of the enzyme suggests that the novel substrate specificity may be explained by substrate interactions with residues Tyr42, His98, and His9, which interact with the hydroxyl groups of C2, C3, and C4, respectively, oriented in the same direction. L-Talose and D-ribulose exhibited the highest activity among the aldoses and ketoses, respectively. Ribose 5-phosphate isomerase catalyzed the conversion of L-talose to L-tagatose with an 89% conversion yield after approximately 90 min, while D-ribulose was converted to D-ribose with a 38% conversion yield.     

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.     

Critical residues for the specificity of cofactors and substrates in human estrogenic 17beta-hydroxysteroid dehydrogenase 1: variants designed from the three-dimensional structure of the enzyme.

Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.