Rational design of L-arabinose isomerase from Lactobacillus fermentum and its application in D-tagatose production / 生物工程学报

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Km and kcat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene / 生物工程学报

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Pentoses Used in Cultures of Synechococcus nidulans and Spirulina paracas: Evaluation of Effects in Growth and in Content of Proteins and Carbohydrates

Abstract The biological assimilation of the sugars present in lignocellulosic residues has gained prominence since these residues are the most abundant and economic residues in nature. Thus, the objective of this work was to determine whether the use of D-xylose and L-arabinose as sources of carbon in Synechococcus nidulans and Spirulina paracas cultures affects the growth and production of proteins and carbohydrates. Kinetic growth parameters, pentose consumption, protein content and carbohydrates were evaluated. Synechococcus nidulans and Spirulina paracas consumed all concentrations of pentose used. The highest cellular concentration (1.37 g.L-1) and the highest protein productivity (54 mg.L-1.d-1) were obtained for Spirulina paracas, which was submitted to the addition of 38.33 mg.L-1 D-xylose and 1.79 mg.L-1 L-arabinose. The use of pentose promoted the accumulation of proteins for the studied microalgae. This is one of the first works to report protein bioaccumulation as a result of pentose addition.

Quality evaluation and discrimination of Polygonati Rhizoma from three origins based on polysaccharide composition and content / 中草药

Objective: To compare the composition and content of polysaccharides in Polygonati Rhizoma by qualitative and quantitative analysis combined with chemometrics, and provide the reference for the quality evaluation of Polygonati Rhizoma. Methods Fourier transform infrared spectroscopy (FTIR) and pre-column derivatization-HPLC (PMP-HPLC) were employed to reveal the differences of polysaccharide among the three species, and the content of total polysaccharide was determined by ultraviolet spectroscopy (UV). Results:The result indicated that the average spectra and the second derivative atlas in FTIR fingerprint of Polygonati Rhizoma was significantly different. The PCA, PLS-DA and HCA analysis provided a further refinement of the method to distinguish three species. Furthermore, the PMP-HPLC showed that the components of monosaccharide and polysaccharides of three species were different. The main common components of the 10 common peaks were identified, which were as follows: D-mannose, D-ribose, L-rhamnose, D-galacturonic acid, D-glucuronic acid, D-galactose, D-glucose, D-xylose, D-arabinose and L-fucose. The content of the total polysaccharide meeted the requirements of Chinese pharmacopoeia 2015 edition Conclusion:The study provided a new reference for quality evaluation of P. Rhizoma.

Determination of Sugar,Fructose,Mannose and Glucose in Shenxiong Glucose Injection by UPLC-MS/MS / 中国实验方剂学杂志

Objective: To establish a method for determining arabinose, mannose, fructopyranose and amylaceum in Shenxiong glucose injection by UPLC-MS/MS, so as to provide the basis for the scientific evaluation of the quality of Shenxiong glucose injection, and lay a foundation for the safe use of drugs in clinic. Method: Domestic GDX-403 solid-phase extraction column was used to purify Waters ACQUITY UPLC BEH Xbridge Amide column (2.1 mm×100 mm, 1.7 μm)at the column temperature of 35℃, and the mobile phase was 0.1% ammonia, 0.1% acetonitrile-0.1% ammonia water and water 85:15. The contents of arabinose, mannose, fructose and glucose in Shenxiong glucose injection were determined by UPLC-MS/MS with a flow rate of 0.3 mL·min-1. Result: A method was established to determine arabinose, mannose, fructopyranose and amylaceum in Shenxiong glucose injection. The concentration range of arabinose, mannose, fructopyranose and amylaceum showed a good linear relationship with the peak area, with a good repeatability and precision. Recoveries were 98.43%, 102.13%, 100.72%, 101.75%, and RSD were 2.4%, 1.3%, 3.1%, 2.7%. Arabinose and mannose content were stable in five batches of Shenxiong glucose injection. Conclusion: The method is simple and specific. Compared with the determination of total sugar, the method is more scientific and stable, and can be used for the quality control of Shenxiong glucose injection.

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

Monosaccharide composition analysis on polysaccharides in Polygonatum cyrtonema by high performance anion-exchange chromatography with pulsed amperometric detection / 中草药

Objective: To develop a method for determination of monosaccharide composition of Polygonatum cyrtonema polysaccharides (PCR) by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC- PAD). Methods: The water-soluble crude polysaccharides were extracted from the P. cyrtonema Hua, then the polysaccharides were hydrolyzed with sulfuric acid, then separated by CarboPac PA1 column (250 mm × 4 mm, 10.0 μm) and with gradient elution, determination of monosaccharide composition in PCP by HPAEC-PAD. Results: The results showed that within the range of 1-100 mg/L it has good linearity (r > 0.999), The detection limitation was 0.015-0.025 mg/L, 1.35%-2.80% RSD, the recovery rates were 88.36%-111.3%. The results showed that the polysaccharides in P. cyrtonema Hua from different regions were composed of rhamnose, arabinose, galactose, glucose, mannose, and fructose. Conclusion: The proposed method has good separation effect and high sensitivity, which can be used for the study on monosaccharide composition and content determination of PCP.

Fermentations of xylose and arabinose by Kluyveromyces marxianus / 生物工程学报

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

Monosaccharide compositions of Codonopsis pilosula polysaccharides and their correlation analysis on cytotoxic activities against HepG2 cells / 中草药

Objective: To study the correlation between monosaccharide compositions and their in vitro cytotoxic activities. Methods: Twenty-six Codonopsis pilosula polysaccharides (CPPs) were extracted by water extraction and alcohol precipitation method from C. pilosula collected from 26 different habitats. Total carbohydrate contents of the polysaccharides were determined by the phenol-sulfuric acid colorimetric method. The methods of m-hydroxydiphenyl and GC were all applied to the determination of the galacturonic acid contents in polysaccharides, and the compositional analysis was performed using the aldononitrile acetate method while the fructose contents was analyzed by the trimethylsilyl ether method. In vitro cytotoxic activities against human hepatocellular carcinoma HepG2 cells of the 26 batches of CPPs were evaluated by MTT. Then, hierarchical clustering analysis (HCA) was used to study the classification of 26 batches of C. pilosula and partial least squares (PLS) was used to investigate the correlation between the monosaccharide compositions and in vitro cytotoxic activities of the polysaccharides. Results: The results showed that all the 26 batches of CPPs possessed the cytotoxic activities against HepG2 cells. The 8th CPPs sample, which was extracted from the C. pilosula collected in Wen county (Gansu province, China), had the highest growth inhibitory rate on HepG2 cells and the inhibitory rate was 36.36%. In addition, the monosaccharide contents of the 26 batches of CPPs were different from each other. The results of HCA showed that the C. pilosula could not be classified according to the monosaccharide compositions. The PLS results indicated that galacturonic acid, arabinose, rhamnose, galactose, and fructose were correlated positively with the cytotoxic activities, while mannose, xylose, and glucose were correlated negatively with the cytotoxic activities. Conclusion: The in vitro cytotoxic activities of CPPs are highly correlative to their monosaccharide compositions, and the CPPs containing much galacturonic acid have the obviously cytotoxic activities.

Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana / 生物工程学报

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 β-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 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ 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.

Efficacy of L-Arabinose for bowel preparation before colonoscopy / 中华消化内镜杂志

Objective To evaluate the efficacy of L?Arabinose for bowel preparation before colonos?copy. Methods A total of 170 patients who underwent colonoscopy were randomized into 2 groups. The ex?perimental group (n=85) used L?Arabinose for bowel preparation, while the control group (n=85) used polyethylene glycol electrolyte solution ( PEG?ELS ) . The degree of comfort, adverse effects, and the visibility during colonoscopy were observed. Results Premedication of L?Arabinose for bowel preparation yielded to more comfort ( U=-4?349,P=0?000) , less adverse effects (χ2=29?27,P=0?000) , and similar visibility during colonoscopy ( U=-0?875,P=0?381) compared with PEG?ELS. Conclusion L?Arabinose is safe, comfortable, and effective for bowel preparation before colonoscopy.

Arabinose-controlled expression of htrA in Shigella flexneri through suicide plasmid-mediated homologous recombination / 军事医学

Objective To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.Methods Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector .The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting .Results The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot .Conclusion Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein .After being induced with arabinose , HtrA protein can be expressed normally .

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using mixed xylose/arabinose as substrate

Background: Biological hydrogen production by microorganisms can be divided into two main categories i.e. photosynthetic organisms that produce hydrogen using light as energy source and anaerobic bacteria that produce hydrogen via dark fermentation. Dark fermentative hydrogen production by anaerobic bacteria has the advantages of a higher HPR without illumination and of the capability to convert various kinds of substrate. Results: Thermophilic hydrogen producer was isolated from elephant dung and identified as Thermoanaerobacterium thermosaccharolyticum KKU-ED1 by 16S rRNA gene analysis, which was further used to produce hydrogen from mixed pentose sugar i.e., xylose/arabinose. The optimum conditions for hydrogen production from mixed xylose/arabinose by KKU-ED1 were a 1:1 xylose/arabinose mixture at the total concentration of 5 g/L, initial pH of 6.5 and temperature of 55ºC. Under the optimum conditions, hydrogen from sugar derived from acid-hydrolyzed sugarcane bagasse at a reducing sugar concentration were achieved. Soluble metabolite product (SMP) was predominantly acetic acid indicating the acetate-type fermentation. Conclusions: The strain KKU-ED1 appeared to be a suitable candidate for thermophilic fermentative hydrogen production from hemicellulosic fraction of lignocellulosic materials due to its ability to use various types of carbon sources.

Inhibitory Effect of Pentose on Biofilm Formation by Oral Bacteria

A number of bacterial species coexist in oral cavities as a biofilm rather than a planktonic arrangement. By forming an oral biofilm with quorum sensing properties, microorganisms can develop a higher pathogenic potential and stronger resistance to the host immune system and antibiotics. Hence, the inhibition of biofilm formation has become a major research issue for the future prevention and treatment of oral diseases. In this study, we investigated the effects of pentose on biofilm formation and phenotypic changes using wild type oral bacteria obtained from healthy human saliva. D-ribose and D-arabinose were found to inhibit biofilm formation, but have no effects on the growth of each oral bacterium tested. Pentoses may thus be good candidate biofilm inhibitors without growth-inhibition activity and be employed for the future prevention or treatment of oral diseases.

Research Progress on the Microbial L-Arabinose Isomerase / 微生物学通报

L-arabinose isomerase (L-AI) can isomerize L-arabinose and D-galactose into L-ribulose and D-tagatose, respectively, which is currently the most effective biological catalyst for D-tagatose production. The crystal structure of L-AI has been solved recently and its gene has been cloned, sequenced and overex- pressed. L-AI improved by protein engineering will be the dominant enzyme for industrial production of D-tagatose. This paper reviewed researches on protein structure and function, properties and application in D-tagatose production of L-AI, and the long-term potential development of L-AI was prospected.

Experimental Study on Effects of Hypoglycemic and Antiobesity of L-Arabinose in New Zealand Rabbit / 中国中医药信息杂志

Objective To research the function of hypoglycemic and antiobesity of L-Arabinose in New Zealand rabbit. Methods Thirty-two New Zealand white rabbits were randomly divided into 4 groups, 8 rabbits in each group. The control group was fed with high fat and high sucrose diet only. L-Arabinose group was fed with high fat and high sucrose diet along with L-Arabinose, the dosage were 0.308 5, 0.617, 0.925 5 g/kg respectively. After fed for two months, all groups were given L-Arabinose, and blood glucose was detected after orally given sucrose 0, 0.5, 2 h. After intervention of L-Arabinose for one month, the data of weight, food intake, stool quantity and the fat index were observed. Results L-Arabinose decreased significantly sucrose absorption, lower the fasting glucose level and the data of area-under-curve (AUC) significantly. Conclusion L-Arabinose proved to be highly effective in preventing the rise of circulating glucose and fat.

Purification and Characterization of L-AI from Bacillus Stearothermophilis IAM 11001 Expressed in E.coli / 中国生物工程杂志

Thermostable L-arabinose isomerase (L-AI) is the most potential enzyme for the biological production of D-tagatose from D-galactose, a novel functional factor. Gene araA encoding the L-arabinose isomerase from Bacillus stearothermophilis IAM 11001 was cloned and expressed in Escherichia coli. The araA gene of 1491 bp has 95% identity with L-AI from Thermus sp. IM6501. The GenBank accession number for the nucleotide sequence of this araA gene determined in this work is EU394214.The bacterium was induced by IPTG and analyzed by SDS-PAGE, approximately 59 kDa exogenous protein was observed on the SDS-PAGE. The recombinant L-AI was purified to electrophoretical homogeneity with affinity chromatography, and the activity of recombinant L-AI was also studied. The bioconversion rate of D-galactose to D-tagatose reached 39.4% after 24h whole cell reaction.