Jianjun Pei

Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose

Backgroundβ-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant β-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications.ResultsThe β-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of β-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel β-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2–7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (Km value of 0.62 mM) and cellobiose (Km value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat for cellobiose was 67.7 s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290 mM. It was activated by glucose at concentrations lower that 200 mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600 mM glucose.ConclusionsThe article provides a useful novel β-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris

AbstarctA laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Metabolic Engineering of Escherichia coli for Astragalin Biosynthesis

Astragalin (kaempferol 3-O-glucoside) is used as a standard to assess the quality of Radix astragali and has exhibited a number of biological properties. In this work, we screened several UDP-dependent glycosyltransferases (UGT) for their potential as efficient biocatalysts for astragalin synthesis. The highest astragalin production with 285 mg/L was detected in the recombinant strain expressing UGT from Arabidopis thaliana (AtUGT78D2). To further improve astragalin production, an efficient UDP-glucose synthesis pathway was reconstructed in the recombinant strain by introducing sucrose permease, sucrose phosphorylase, and uridylyltransferase. On the basis of those results, a recombinant strain, BL21-II, was constructed to produce astragalin. By optimizing conversion conditions, astragalin production was increased from 570 to 1708 mg/L. The production was scaled up using a fed-batch fermentation, and maximal astragalin production was 3600 mg/L, with a specific productivity of 150 mg/L/h after 24 h incubation and a corresponding molar conversion of 91.9%, the highest yield reported to date.

Characterization of a novel arabinose-tolerant α-L-arabinofuranosidase with high ginsenoside Rc to ginsenoside Rd bioconversion productivity.

(i) To investigate the enzymatic characterization of α‐l‐arabinofuranosidase from Thermotoga thermarum DSM5069. (ii) To evaluate the performance of its excellent properties on converting ginsenoside Rc to ginsenoside Rd.

RNA-Seq analysis and comparison of the enzymes involved in ionone synthesis of three cultivars of Osmanthus

Abstract To comprehend the molecular mechanisms that control the differences in the composition of Osmanthus essential oils, the RNA-seq data and differentially expressed genes in different cultivar Osmanthus were studied. cDNA libraries of “jinqiugui,” “baijie,” and “rixianggui” were sequenced using Illumina HiSeq TM 2000. All of the enzymes involved in ionone synthesis were verified. DEGs were revealed and their enriched pathways were analyzed. A total of 20 DEGsencoding four enzymes that were potential candidates involved in ionone biosynthesis, as well as ispH, GPPS, ZDS, and CCD. It provided a way for Osmanthus oil monomer material to be synthesized in vitro.

Data on thermostable β-glucosidase immobilized by Zn 2+

In this article, the methods for detection of enzyme activity and protein concentration are described. The data of the calibration curves can be used for a further understanding on the assays of enzyme activity measured with p-nitrophenyl-β-D-glucopyranoside (pNPG) or cellobiose as the substrate. In addition, the data presented provides an analytic method for measuring protein concentration in mixed samples.

Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C

In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl β-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0-8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s-1 and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.

Biotransformation of Ginsenosides Re and Rg1 into Rg2 and Rh1 by Thermostable β-Glucosidase from Thermotoga thermarum

The recombinant thermostable β-glucosidase from Thermotoga thermarum DSM 5069T exhibited high selectivity to catalyze the conversion of ginsenoside Re and Rg1 to the more pharmacologically active minor ginsenoside Rg2 and Rh1, respectively. At a concentration of 1.36 U/mL of the enzyme, a temperature of 85°C, and pH 5.5, 10 g/L ginsenoside Re was transformed into 8.02 g/L Rg2 within 60 min, and 2 g/L ginsenoside Rg1 was transformed into 1.56 g/L Rh1 within 60 min. This paper provides the first report on the production of ginsenoside Rg2 and Rh1 by a highly thermostable β-glucosidase.

Characterization flavanone 3β-hydroxylase expressed from Populus euphratica in Escherichia coli and its application in dihydroflavonol production

Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe2+ and Fe3+. The KM and Vmax for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The Km and Vmax for eriodictyol were 0.18 mM and 0.013 μmoles mg–1min–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD600 of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.