Ye-Wang Zhang

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM.

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and beta-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.

Preparation Fe3O4@chitosan magnetic particles for covalent immobilization of lipase from Thermomyces lanuginosus.

Magnetic Fe3O4@chitosan nanoparticles were prepared by a simple in situ co-precipitation method and characterized by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). The prepared Fe3O4@chitosan nanoparticles were used for covalent immobilization of lipase from Thermomyces lanuginosus by chemical conjugation after electrostatic entrapment (CCEE). The optimal immobilization conditions were obtained as follows: enzyme/support 19.8 mg/g, pH 5.0, time 4h and temperature 30 °C. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 16.8 mg/g-support were obtained. Broad pH tolerance and high thermostability could be achieved by immobilization. The immobilized lipase retained 70% initial activity after ten cycles. Kinetic parameters Vmax and Km of free and immobilized lipase were determined as 5.72 mM/min, 2.26 mM/min and 21.25 mM, 28.73 mM, respectively. Ascorbyl palmitate synthesis with immobilized lipase was carried out in tert-butanol at 50 °C, and the conversion of ascorbic acid was obtained higher than 50%. These results showed that the immobilization of lipase onto magnetic chitosan nanoparticles by the method of CCEE is an efficient and simple way for preparation of stable lipase.

Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus.

NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ~50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration.

Spectral imaging of single molecules by transmission grating-based epi-fluorescence microscopy.

A spectral imaging method of single protein molecules labeled with a single fluorophore is presented. The method is based on a transmission grating and a routine fluorescence microscope. The bovine serum alubmin (BSA) and antiBSA molecules labeled with Alexa Fluor 488 and Alexa Fluor 594, respectively, are used as the model proteins. The fluorescence of single molecules is dispersed into zeroth-order spectrum and first-order spectrum by the transmission grating. Results show that the fluorescence emission spectrum of single molecule converted from the first-order spectral imaging is in good agreement with the bulk fluorescence spectrum. The spectral resolution of 2.4nm/pixel is obtained, which is sufficient for identifying the molecular species in a multicomponent system.

Preparation of Optically Pure tert‐Leucine by Penicillin G Acylase‐Catalyzed Resolution

Abstract Penicillin G acylase, from Kluyvera citrophila, was used in kinetic resolution of DL‐ tert‐leucine. N‐phenylacetylated‐DL‐tert‐leucine, chemically synthesized from DL‐ tert‐leucine, was enantioselctively hydrolyzed by penicillin G acylase to obtain L‐tert‐leucine, D‐ tert‐leucine was prepared by acid‐catalyzed hydrolysis of the remaining substrate. The total yields of D‐ tert‐leucine and L‐tert‐leucine are 80.6% and 83.1%, respectively. The enantiomeric excess of the two products, D‐ tert‐leucine and L‐tert‐leucine, are 98.5% and 99%. This is a practical way for the preparation of D‐ tert‐leucine and L‐tert‐leucine.

Immobilization of dehydrogenase onto epoxy-functionalized nanoparticles for synthesis of (R)-mandelic acid.

Epoxy functionalized magnetic Fe3O4@SiO2 nanoparticles were successfully prepared and characterized by Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The prepared nanoparticles were used for immobilization of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae by covalent attachment. The optimal immobilization conditions were obtained as follows: enzyme/support 4.49mg/g, pH 8.0, buffer concentration 0.05M, time 12h and temperature 30°C. Under these conditions, a high immobilization yield and efficiency of above 92% were obtained after the optimization. Broad pH tolerance and high thermostability were achieved by the immobilization. The immobilized ADH retained about 84% initial activity after five cycles. Kinetic parameters Vmax and Km of free and immobilized ADH were determined as 56.72μM/min, 44.27μM/min and 11.54mM, 31.32mM, respectively. (R)-mandelic acid synthesis with the immobilized ADH was carried out, and the yield of (R)-mandelic acid was as high as 64%. These results indicate that the ADH immobilized onto epoxy-functionalized nanoparticles is an efficient and simple way for preparation of stable ADH, and the immobilized ADH has potential applications in the production of (R)-mandelic acid.

Immobilization of Bacillus licheniformis L-arabinose isomerase for semi-continuous L-ribulose production.

Bacillus licheniformis L-arabinose isomerase (BLAI) with a broad pH range, high substrate specificity, and high catalytic efficiency for L-arabinose was immobilized on various supports. Eupergit C, activated-carboxymethylcellulose, CNBr-activated agarose, chitosan, and alginate were tested as supports, and Eupergit C was selected as the most effective. After determination of the optimum enzyme concentration, the effects of pH and temperature were investigated using a response surface methodology. The immobilized BLAI enzyme retained 86.4% of the activity of the free enzyme. The optimal pH for the immobilized BLAI was 8.0, and immobilization improved the optimal temperature from 50 °C (free enzyme) to a range between 55 and 65 °C. The half life improved from 2 at 50 °C to 212 h at 55 °C following immobilization. The immobilized BLAI was used for semi-continuous production of L-ribulose. After 8 batch cycles, 95.1% of the BLAI activity was retained. This simple immobilization procedure and the high stability of the final immobilized BLAI on Eupergit C provide a promising solution for large-scale production of L-ribulose from an inexpensive L-arabinose precursor.

Secretory Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin

Abstract In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K/ILP. Pichia pastoris GS115 was transformed by pPIC9K/ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g/L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U/mg.

Preparation, characterization and antibacterial activity of octenyl succinic anhydride modified inulin

Octenyl succinic anhydride modified inulin (In-OSA) was synthesized via chemical modification of inulin with octenyl succinic anhydride (OSA). The esterification of inulin with OSA was confirmed by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and degree of substitution (DS) calculation. Antibacterial activity of In-OSA against Staphylococcus aureus and Escherichia coli was investigated by minimum inhibitory concentration (MIC) and inhibition rate determination. The results showed that inhibition rates against both E.coli and S. aureus increased with the increase of the In-OSA concentration. And the MICs against E. coli and S. aureus were 1% and 0.5% (w/v), respectively. The antibacterial mechanism was analyzed with the results of the proteins and nucleic acids leakage, SEM and negative staining transmission electron microscopy (TEM). Both the leakages of proteins and nucleic acids increased with the increase of the In-OSA concentration. The leakage occurred mainly in the early stage which indicated that cell membrane and wall were destroyed by In-OSA quickly. The images of SEM and negative staining TEM suggested that the cell membranes and cell walls of S. aureus were damaged more severely and even destroyed completely; but only pores appeared on the surface of E. coli.

One-pot, two-step enzymatic synthesis of amoxicillin by complexing with Zn2+

A one-pot, two-step enzymatic synthesis of amoxicillin from penicillin G, using penicillin acylase, is presented. Immobilized penicillin acylase from Kluyvera citrophila was selected as the biocatalyst for its good pH stability and selectivity. Hydrolysis of penicillin G and synthesis of amoxicillin from the 6-aminopenicillanic acid formed and d-p-hydroxyphenylglycine methyl ester were catalyzed in situ by a single enzyme. Zinc ions can react with amoxicillin to form complexes, and the yield of 76.5% was obtained after optimization. In the combined one-pot synthesis process, zinc sulfate was added to remove produced amoxicillin as complex for shifting the equilibrium to the product in the second step. By controlling the conditions in two separated steps, the conversion of the first and second step was 93.8% and 76.2%, respectively. With one-pot continuous procedure, a 71.5% amoxicillin yield using penicillin G was obtained.