RESUMO
Recently, production of D-mannose becomes a hotspot owing to it exhibiting many physiological functions on people's health and wide applications in food and pharmaceutical field. The use of biological enzymes to production of D-mannose is of particular receiving considerable concerns due to it possessing many merits over chemical synthesis and plant extraction strategies. D-Lyxose isomerase (D-LIase) plays a pivotal role in preparation of D-mannose from d-fructose through isomerization reaction. Thus, a novel putative D-LIase from thermophiles strain Thermoprotei archaeon which was expressed in E. coli BL21(DE3) was first identified and biochemically characterized. The recombinant D-LIase showed an optimal temperature of 80 and 85 °C and pH of 6.5. It was highly thermostable at 70 °C and 80 °C after incubating for 48 h and 33 h, respectively, with retaining over 50% of the initial activity. A lower concentration of Ni2+ (0.5 mM) could greatly increase the activity by 25-fold, which was rare reported in other D-LIases. It was a dimer structure with melting temperature of 88.3 °C. Under the optimal conditions, 15.8 g L-1 of D-mannose and 33.8 g L-1 of D-xylulose were produced from 80 g L-1 of d-fructose and D-lyxose, respectively. This work provided a promising candidate sugar isomerase T. archaeon D-LIase for the production of D-mannose and D-xylulose.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Archaea/enzimologia , Níquel/química , Proteínas Recombinantes/biossíntese , Soluções Tampão , Clonagem Molecular , Cristalização , Escherichia coli/metabolismo , Frutose/química , Concentração de Íons de Hidrogênio , Íons , Cinética , Manose/química , Peso Molecular , Pentoses/química , Fosfatos , Filogenia , Especificidade por Substrato , TemperaturaRESUMO
A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.
Assuntos
Aldose-Cetose Isomerases , Proteínas de Bactérias , Expressão Gênica , Geobacillus stearothermophilus/genética , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7 H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.05, designed with one pulse feed (t = 7 H) followed by a continuous feed and (ii) 2PMF -0.05, designed with two consecutive pulse feeds (t = 7 and 10 H) followed by a continuous feed. The continuous feeding of molasses for both fermentation strategies employed the same precalculated feeding rate, µo = 0.05 H-1 . The maximum r-GI activities exhibited by PM-0.05 and 2PMF -0.05 were 29,050 and 30,642 U dm-3 , respectively. On the one hand, compared to PM-0.05 r-GI activity reached its maximum within a shorter cultivation time (∆tmax = 2 H) at 2PMF -0.05, which could be preferable in terms of manufacturing costs and possible risks; on the other hand, PM-0.05 is a simpler fermentation regime compared to 2PMF -0.05 with respect to manipulations that should be considered in large-scale production.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Beta vulgaris/metabolismo , Escherichia coli/metabolismo , Reatores Biológicos , Estabilidade Enzimática , Escherichia coli/citologia , Hidrólise , Proteínas Recombinantes/biossíntese , Thermus thermophilus/enzimologiaRESUMO
d-Mannose isomerase (MIase) catalyzes the conversion of d-fructose to d-mannose. In this study, the MIase encoding gene (yihS) from Escherichia coli BL21 contains an ORF of 1242bp, was cloned and expressed in Bacillus subtilis WB800. This heterologous expression resulted in a hexamer with a molecular weight of 274.5kDa and Tm of 61.4°C. Efficient MIase secretory expression by the robust recombinant B. subtilis was achieved with activity of 51.2U/ml (d-mannose forming). Its optimal temperature and pH were 45.0°C and 7.0, respectively. Using d-fructose as the substrate, Km, kcat and catalytic efficiency value of kinetic reaction were 203.7±6.7mM, 27.7±0.7s(-1) and 136.0±2.9M(-1)s(-1), respectively. The production of d-mannose reached about 150g/l with approximately 25% turnover yield under the optimum conditions. These results demonstrated that B. subtilis was a promising candidate of MIase expression system for d-mannose production.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Bacillus subtilis/genética , Proteínas de Escherichia coli/biossíntese , Escherichia coli/química , Frutose/metabolismo , Manose/biossíntese , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Bacillus subtilis/enzimologia , Clonagem Molecular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Fases de Leitura Aberta , Multimerização Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , TemperaturaRESUMO
Saccharomyces cerevisiae strains with xylose isomerase (XI) pathway were constructed using a flocculating industrial strain (YC-8) as the host. Both strains expressing wild-type xylA (coding XI) from the fungus Orpinomyces sp. and the bacterium Prevotella ruminicola, respectively, showed better growth ability and fermentation capacity when using xylose as the sole sugar than most of the reported strains expressing XI. Codon optimization of both XIs did not improve the xylose fermentation ability of the strains. Adaption significantly increased XI activity resulting in improved growth and fermentation. The strains expressing codon-optimized XI showed a higher increase in xylose consumption and ethanol production compared to strains expressing wild XI. Among all strains, the adapted strain YCPA2E expressing XI from P. ruminicola showed the best performance in the fermentation of xylose to ethanol. After 48 h of fermentation, YCPA2E assimilated 16.95 g/L xylose and produced 6.98 g/L ethanol. These results indicate that YC-8 is a suitable host strain for XI expression, especially for the codon-optimized XI originating from P. ruminicola.
Assuntos
Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Reatores Biológicos , Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aldose-Cetose Isomerases/biossíntese , Códon/genética , Etanol/provisão & distribuição , Floculação , Neocallimastigales/enzimologia , Neocallimastigales/genética , Prevotella ruminicola/enzimologia , Prevotella ruminicola/genética , Xilose/metabolismoRESUMO
Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 â, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²âº and Cd²âº metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Proteínas de Arabidopsis/biossíntese , Arabidopsis/enzimologia , Clonagem Molecular , Escherichia coli/metabolismo , Metais , Pentosefosfatos , Proteínas Recombinantes/biossínteseRESUMO
Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.
Assuntos
Engenharia Metabólica/métodos , Synechocystis , Xilose/metabolismo , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Synechocystis/genética , Synechocystis/metabolismo , Xilose/genéticaRESUMO
The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L(-1) at t = 66 h and 32530 U L(-1) at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Códon , Pichia/enzimologia , Oxirredutases do Álcool/química , Aldose-Cetose Isomerases/genética , Sequência de Bases , Reatores Biológicos , Carbono/química , Fermentação , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Manitol/química , Metanol/química , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Sorbitol/química , Espectrofotometria Ultravioleta , Temperatura , Thermus thermophilus/enzimologiaRESUMO
Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.
Assuntos
Abietanos/biossíntese , Aldose-Cetose Isomerases/genética , Hidroximetilglutaril-CoA Redutases/genética , Salvia miltiorrhiza/genética , Abietanos/química , Aldose-Cetose Isomerases/biossíntese , Compostos de Bifenilo/química , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/metabolismo , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/metabolismo , Expressão Gênica , Hidroximetilglutaril-CoA Redutases/biossíntese , Oxirredução , Picratos/química , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Salvia miltiorrhiza/enzimologiaRESUMO
Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Assuntos
Acanthamoeba castellanii/enzimologia , Aldose-Cetose Isomerases/biossíntese , Amebíase/patologia , Parede Celular/metabolismo , Glucosiltransferases/biossíntese , Acanthamoeba castellanii/genética , Acanthamoeba castellanii/metabolismo , Benzenossulfonatos , Parede Celular/química , Parede Celular/genética , Celulose/biossíntese , Regulação para Baixo , Encefalite/parasitologia , Glucosiltransferases/genética , Ceratite/parasitologia , Microscopia Eletrônica de Transmissão , Interferência de RNA , RNA Interferente PequenoRESUMO
Glucose isomerase (GIase) catalyzes the isomerization of D-glucose to D-fructose. The GIase from Thermobifida fusca WSH03-11 was expressed in Escherichia coli BL21(DE3), and the purified enzyme took the form of a tetramer in solution and displayed a pI value of 5.05. The temperature optimum of GIase was 80 °C and its half life was about 2 h at 80 °C or 15 h at 70 °C. The pH optimum of GIase was 10 and the enzyme retained 95 % activity over the pH range of 5-10 after incubating at 4 °C for 24 h. Kinetic studies showed that the K m and K cat values of the enzyme are 197 mM and 1,688 min(-1), respectively. The maximum conversion yield of glucose (45 %, w/v) to fructose of the enzyme was 53 % at pH 7.5 and 70 °C. The present study provides the basis for the industrial application of recombinant T. fusca GIase in the production of high fructose syrup.
Assuntos
Actinomycetales , Aldose-Cetose Isomerases , Proteínas de Bactérias , Expressão Gênica , Proteínas Recombinantes , Actinomycetales/enzimologia , Actinomycetales/genética , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Arabidopsis/enzimologia , Etanol/metabolismo , Flammulina/enzimologia , Flammulina/metabolismo , Aldose-Cetose Isomerases/genética , Arabidopsis/genética , Fermentação , Flammulina/genética , Perfilação da Expressão Gênica , RNA Fúngico/análise , RNA Fúngico/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Xilose/metabolismoRESUMO
Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs) and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Entamoeba histolytica/enzimologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Protozoários/biossíntese , Interferência de RNA , Trofozoítos/enzimologia , Aldose-Cetose Isomerases/genética , Quitina/biossíntese , Entamoeba histolytica/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Proteínas de Protozoários/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-D-xylulose 5-phosphate synthase (dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.
Assuntos
Butadienos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hemiterpenos/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Pentanos/metabolismo , Proteínas de Plantas/biossíntese , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Isomerases de Ligação Dupla Carbono-Carbono/biossíntese , Isomerases de Ligação Dupla Carbono-Carbono/genética , Óperon , Proteínas de Plantas/genética , Populus/enzimologia , Populus/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transferases/biossíntese , Transferases/genéticaRESUMO
L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Hexoses/metabolismo , Pediococcus/genética , Proteínas Recombinantes/biossíntese , Biotransformação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/metabolismo , Vetores Genéticos/genética , Pediococcus/classificação , Pediococcus/isolamento & purificação , Proteínas Recombinantes/genéticaRESUMO
Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 °C, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.
Assuntos
Aldose-Cetose Isomerases/isolamento & purificação , Aldose-Cetose Isomerases/metabolismo , Bifidobacterium/enzimologia , Bifidobacterium/genética , Lactococcus lactis/genética , Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Clonagem Molecular , Expressão Gênica , Hexoses/biossíntese , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Pentoses/biossíntese , TemperaturaRESUMO
The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Thermotoga maritima/enzimologia , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Escherichia coli , Expressão Gênica , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.
Assuntos
Aldose-Cetose Isomerases/biossíntese , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Catálise , Estabilidade Enzimática , Escherichia coli/genética , Hexoses/biossíntese , Recombinação Homóloga , Temperatura Alta , Manganês/farmacologia , Metais/farmacologia , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/metabolismo , EdulcorantesRESUMO
Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.
Assuntos
Arabinose/metabolismo , Clostridium acetobutylicum/enzimologia , Glucose/metabolismo , Redes e Vias Metabólicas/genética , Organismos Geneticamente Modificados/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/deficiência , Xilose/metabolismo , Acetona/metabolismo , Aldose-Cetose Isomerases/biossíntese , Butanóis/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Etanol/metabolismo , Fermentação , Expressão Gênica , Técnicas de Inativação de Genes , Lignina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Simportadores/biossínteseRESUMO
Styrene is a large volume, commodity petrochemical with diverse commercial applications, including as a monomer building-block for the synthesis of many useful polymers. Here we demonstrate how, through the de novo design and development of a novel metabolic pathway, styrene can alternatively be synthesized from renewable substrates such as glucose. The conversion of endogenously synthesized l-phenylalanine to styrene was achieved by the co-expression of phenylalanine ammonia lyase and trans-cinnamate decarboxylase. Candidate isoenzymes for each step were screened from bacterial, yeast, and plant genetic sources. Finally, over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae (originally classified as ferulate decarboxylase) in an l-phenylalanine over-producing Escherichia coli host led to the accumulation of up to 260 mg/L in shake flask cultures. Achievable titers already approach the styrene toxicity threshold (determined as ~300 mg/L). To the best of our knowledge, this is the first report of microbial styrene production from sustainable feedstocks.