High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.     

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana

Escherichia coli cells expressing l-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t _1/2 = 43 days at 70°C) in a continuous recycling mode at 70°C produced 49 and 38 g d-tagatose/l from 180 and 90 g d-galactose/l, respectively, within 12 h.     

Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.     

Preparation of L-arabinose isomerase originated from Escherichia coli as a biocatalyst for D-tagatose production

l-Arabinose isomerase for tagatose production from recombinant Escherichia coli was partially purified 15-fold with a specific activity of 70 U mg^–1 protein. The purified enzyme had a major band when it was subjected to SDS/PAGE. With the purified l-arabinose isomerase, 17.7 g tagatose l^–1 was produced from 50 g galactose l^–1 in 168 h which corresponds to a 34% equilibrium.     

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.     

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%。     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

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.     

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii

The ability to convert d-galactose into d-tagatose was compared among a number of bacterial l-arabinose isomerases (araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis–Menten constants of the enzyme determined with l-arabinose, d-galactose and d-fucose also indicated that this enzyme is an unusual, versatile l-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of d-tagatose at 65 °C. Starting from a 30% solution of d-galactose, the yield of d-tagatose was 42% and no sugars other than d-tagatose and d-galactose were detected. Direct conversion of lactose to d-tagatose in a single reactor was demonstrated using a thermostable -galactosidase together with the thermostable l-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.     

Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501

A gene (thaI) corresponding to l-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the l-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60°C at pH 8 for 30 min of reaction. Zn²⁺ and Ni²⁺ inactivated the catalytic activity of ThaI, 5 mM Mn²⁺ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from d-galactose to d-tagatose was obtained by recombinant ThaI at 60°C over 3 d.     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

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.     

用毕赤酵母的GAP启动子调控表达L-阿拉伯糖异构酶

应用PCR从大肠杆菌基因组中扩增L-阿拉伯糖异构酶基因,用EcoR I和Not I双酶切将其克隆进P.pastoris表达载体,获得重组表达载体pGAP9K-L-ai。通过电转法将pGAP9K—L-ai转化毕赤酵母GS115,筛选高G418抗性和高表达L-阿拉伯糖异构酶的重组工程菌。用葡萄糖作为碳源在摇瓶中发酵48 h,表达重组L-ai 53 mg/L。用毕赤酵母的GAP启动子调控表达的重组L-ai具有异构D-半乳糖生成D-塔格糖的生物学活性。     

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO₄) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO₄ 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.     

Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway

The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.     

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.     

L-arabinose isomerase formation in a conditional mutant of gene araA of Escherichia coli B-r

A temperature-sensitive mutant of Escherichia coli in which the synthesis of l-arabinose isomerase is blocked during growth at 42 C was found to possess the following properties. (i) The mutation occurred in the structural gene for the isomerase, gene araA. (ii) During growth at elevated temperatures the mutant accumulates a product which is a precursor to the active enzyme. (iii) The precursor produced at 42 C is slowly converted to active enzyme at 28 C in the absence of protein and ribonucleic acid synthesis. It is concluded that the mutation results in a change in the structure of isomerase which causes formation of active enzyme to be thermolabile at a step beyond the level of translation.     

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.