Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity

Exploring excellent new pullulanase genes, and enriching pullulanase theory are of great importance to realize the industrialization of pullulanase. Three genes, pulA, pulB and pulC, encoding pullulanases, were cloned from Bacillus cereus GXBC-3 by bioinformatics analyzing the open reading frame in Bacillus cereus, annotated as putative I and II pullulanases in the GenBank database. Characteristics of these recombinant enzymes were inducible intracellular expressed in Escherichia coli, the results showed PulA was typical II pullulanase. Recombinant PulA could hydrolyze alpha-1,4- and alpha-1,6-glycosidic bonds. Its specific activity was 32.89 U/mg with an optimum temperature of 40 degrees C and optimum pH 6.5 using pullulan as substrate. And for soluble starch substrate, its specific activity was 25.71 U/mg with an optimum temperature of 50 degrees C and optimum pH 7.0. PulB and PulC were I pullulanases and only hydrolyzed alpha-1,6-glycosidic bond. The specific activities, optimum temperature and optimum pH of PulB and PulC for pullulan substrate were 228.54 U/mg, 45 degrees C, 7.0 and 229.65 U/mg, 45 degrees C, 6.5, respectively.
Bacillus cereus ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; Glucans ; metabolism ; Glycoside Hydrolases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

We optimized the conditions of simultaneous saccharification and fermentation (SSF) from cassava flour into high-concentration ethanol by thermophilic yeast GXASY-10. Based on the single factor experiment, we screened the important parameters by Plackeet-burman design. We used the path of steepest ascent to approach to the biggest region of ethanol production subsequently. Then, we obtained the optimum values of the parameters by Box-Behnken design. The results showed that the important parameters were the liquefaction time, glucosidase dosages and initial concentration of cassava flour (substrate). The optimum technical conditions were as follows: liquefaction time 35 min, glucosidase dosages 1.21 AGU/g substrate and initial substrate concentration 37.62%. Under such optimum conditions, the ethanol yield of 20 L fermentor reached 16.07% (V/W) after 48 h fermentation at 37 degrees C and 100 r/min. The ethanol content increased 33% than that under the original condition.
Ethanol ; analysis ; metabolism ; Fermentation ; Glucosidases ; pharmacology ; Hot Temperature ; Manihot ; metabolism ; Substrate Specificity ; Yeasts ; physiology

The regions suitable for growing cassava include five provinces in Southern China, with Guangxi alone accounting for over 65% of the total cassava production in the country. In this article, the state-of-the-art development of fuel ethanol production from cassava in China is illustrated by the construction of the cassava fuel ethanol plant with its annual production capacity of 200 000 metric tons. And in the meantime, problems and challenges encountered in the development of China's cassava fuel ethanol are highlighted and the strategies to address them are proposed.
Biofuels ; China ; Conservation of Energy Resources ; Ethanol ; metabolism ; Manihot ; metabolism

Functional improvement to one component of the cellulase, endo-beta-1, 4-glucanase, has been a focus of the recent research in this area. We report here the saturation mutagenesis of the active site of an endoglucanase (CfEG) from termite Coptotermes formosanus. First, three dimensional structure of CfEG was built via homology modeling by using a close-related (79% homology in sequence) endo-beta-1,4-glucanase (NtEG PDB id = 1ks8) from higher termite Nasutitermes takasagoensis as a template. Second, we identified three corresponding amino acid positions at the active site of CfEG by structural superposition onto NtEG. These three putative amino acids for the active site of CfEG, i.e., Asp53, Asp56 and Glu411, were subjected to saturation mutagenesis using degenerate primers. Among the mutants, Asp53Glu and Asp56Cys showed somewhow higher activities than the wildtype, with the latter having more than 3-fold decrease in Km. Double mutation Asp53Leu/Asp56IIe showed nearly 2-fold increase in specific activity and in the same time 2-fold decrease in Km. Saturation mutagenesis to the position Glu411 produced no active mutant, even changing Glu411 explicitly into its similar amino acids such as Glu411Asp and Glu411Gln could not result in any active mutant. These imply that position Glu411 could be extremely important and therefore indispensable for CfEG functionality.
Amino Acids ; genetics ; Animals ; Cellulase ; chemistry ; genetics ; metabolism ; Isoptera ; enzymology ; Mutagenesis, Site-Directed ; Mutation

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.
Antibodies, Bispecific ; biosynthesis ; genetics ; Antibodies, Neoplasm ; biosynthesis ; genetics ; Base Sequence ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; HeLa Cells ; Humans ; Melanoma ; pathology ; Molecular Sequence Data ; Receptors, IgG ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics

By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Antibodies ; genetics ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Interleukin-2 ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics

We optimized the conditions of mixed fermentation of very high gravity ethanol with cassava flour and sugarcane juice. Based on the single factor experiment, we screened the important parameters for very high gravity ethanol fermentation with cassava flour and sugarcane juice by the Plackeet-burman design. Then, we obtained the optimum values of the important parameters by the orthogonal experiments: the mixing ratio of cassava flour to sugarcane juice, 1:5; initial pH of fermentation, 4.0-4.5; the concentrations of urea and MgSO4, 0.25% and 0.04% (W/W), respectively. Finally, we used a gradient temperature control strategy with the optimized conditions, and ethanol concentration of 17.84% (V/V) and fermentation efficiency of 91.82% were achieved, correspondingly.
Biofuels ; analysis ; Ethanol ; analysis ; metabolism ; Fermentation ; Hydrogen-Ion Concentration ; Manihot ; metabolism ; Powders ; Saccharum ; metabolism

Objective To investigate the design,incisional method and clinical experiences of using the mi cro-dissected polyfoliate anterolateral thigh perforator flap to repair of complex soft tissue defect in extremities.Methods From June,2017 to September,2018,12 cases of different kinds of complex soft tissue defect in extremities were repaired by micro-dissected free polyfoliate anterolateral thigh perforator flap.Each flap was divided into two cutaneous perforators to give two separate flap with a common vascular supply.The flaps were cut from the superficial layer of the deep cervical fesciae and without fascia lata.The donor sites were treated by subcutaneous cosmetic suture.Patients were followed-up by outpatient service,telephone and WeChat video to observe and record the flap's appearance,sensory recovery,extremities function and the scars of the donor site to evaluate its clinical efficacy.Results All flaps survived without vascular crisis happened except one-leaf necrosis occurred,which healed with local flap transferring.The donor sites remained linear scars.The mean flap thickness of this group after micro-dissection was (4.5±0.5) mm.All the patients were followed-up for 5-15 months.The 2 point discrimination ranged between 0.5-2.0 cm.Sensory restoration ranking was S3-S3+.The range of montion of wrist joint was 65°-90°,and that of ankle joint was 40°-60°.Conclusion The micro-dissected polyfoliate anterolateral thigh perforator flap is an ideal method for complex and irregular multiple sites soft tissue defect in extremities as it can keep good economic benefit and minimal damage to the donor site.

Exploring new beta-glucosidase genes is of great importance to industrialize beta-glucosidase. The genomes of Aspergillus fumigatus contain a bgl gene, which encodes a 65 kDa putative beta-glucosidase. The bgl gene was cloned into an expression plasmid and transformed to Escherichia coli BL21 (DE3). The bgl was expressed upon induction of Isopropyl beta-D-1-thiogalactopyranoside (IPTG). The recombinant protein was purified by GST-tag affinity chromatography. The purified recombinant Bgl was characterized using Esculin as substrate. The optimum temperature and pH were 45 degrees C and 5.0-6.0, respectively. The K(m) for Esculin was 17.7 mmol/L. The enzyme was stable in the range of pH 4-7. After incubation at 70 degrees C for 2 h, the recombinant Bgl remained 60% of its activity. Metal ions and chemical reagents had different influences on the activity of beta-glucosidase. Ca2+ (1 mmol/L) could increase enzyme activity slightly. On the contrary, the enzyme activity was greatly inhibited by 5 mmol/L Sodium dodecyl sulfate (SDS). Based on our results, the A. fumigatus Bgl was thermostable beta-glucosidase.
Aspergillus fumigatus ; enzymology ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; beta-Glucosidase ; biosynthesis ; genetics ; metabolism