Yushu Ma

Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production.

A lipase-producing bacterium K107 was isolated from soil samples of China and identified to be a strain of Proteus sp. With genome-walking method, the open reading frame of lipase gene lipK107, encoding 287 amino acids, was cloned and expressed in a heterologous host, Escherichia coli BL21 (DE3). The recombinant lipase was purified and characterized, and the optimum pH of the purified LipK107 was 9, at 35 degrees C. The recombinant E. coli expressing lipK107 was applied in biodiesel production in the form of whole-cell biocatalyst. Activity of the biocatalyst increased significantly when cells were permeabilized with 0.3% (w/v) cetyl-trimethylammoniumbromide (CTAB). This transesterification was carried out efficiently in a mixture containing 5M equivalents of methanol to the oil and 100% water by weight of the substrate. It was the first time to use E. coli whole-cell biocatalyst expressing lipase in biodiesel production, and the biodiesel reached a yield of nearly 100% after 12h reaction at the optimal temperature of 15 degrees C, which was the lowest temperature among all the known catalyst in biodiesel production.

Mutant Library Construction in Directed Molecular Evolution

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).

Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface

BackgroundFor industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.ResultsTwo Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.ConclusionThe LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Structure-Based Stabilization of an Enzyme: The Case of Penicillin Acylase from Alcaligenes faecalis

The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.

The transduction of His-TAT-p53 fusion protein into the human osteogenic sarcoma cell line (Saos-2) and its influence on cell cycle arrest and apoptosis

The p53 gene is a tumor suppressor gene. It encodes a nuclear phosphoprotein p53 involved in the regulation of cell cycle arrest and apoptosis to maintain the genomic integrity of the cell. As mutations of p53 gene are found in most human cancers, p53 protein becomes a hot target in the research of anticancer therapy. In the present study, an 11-amino acid domain of TAT protein which has been demonstrated to be able to transduce across cell membranes was fused with p53. The result revealed that the fusion protein His-TAT-p53 accumulated in the nucleus and inhibited the growth of the Saos-2 cells. Besides apoptosis, an increased percentage of G2 phase suggested that the transduction of His-TAT-p53 into cells might be associated with a G2 arrest of cell cycle.

Enhancing enzymatic activity of penicillin G acylase by coexpressing pcm gene.

Penicillin G acylase (PGA; E.C. 3.5.1.11) is an important enzyme which has broad applications in industries of β-lactim antibiotics production. In this study, a promising PGA gene from Alcaligenes faecalis (afpga) and another pcm gene encoding protein isoaspartate methyltransferase (PIMT) were constructed into pET43.1a(+) and pET28a(+), respectively. The recombinant plasmids pETAFPGA and pETPCM were transformed into the same host cell Escherichia coli BL21 (DE3). Results suggested that the two plasmids could peacefully exist in the host cell and the two genes could be efficiently expressed after induction. The product of pcm gene could function as a helper molecule for enzyme AFPGA. PIMT increased the enzymatic activities in supernatant of ferment broth (1.6 folds) and cell lysate (1.8 folds), while it did not significantly affect the expression level of penicillin G acylase.

Expression and Purification of Exendin-4 Dimer in Escherichia coli and Its Interaction with GLP-1 Receptor In Vitro

Exendin-4 is a 39 amino acid peptide isolated from the Gila monster salivary gland. It is 53% homologous to GLP-1 and exhibits similar glucoregulatory activities. In this study, exendin-4 dimer (D-Ex4) was constructed, cloned into plasmid pET32a(+) and expressed in E. coli BL21(DE3). The fusion protein with His-tag at the N-terminus was purified with a Ni-NTA-agarose column. After proteolytic cleavage, D-Ex4 peptide with high purity was obtained by HPLC. The results obtained by chemical cross-linking showed that D-Ex4 maintained affinity to GLP-1 receptor.

Screening of a Phage Display Library of Exendin-4 Mutants with the Extracellular Domain of Rat GLP-1 Receptor

A phage display library of exendin-4 mutants was screened with the extracellular domain of rat glucagon-like peptide 1 receptor as the target. A novel variant of exendin-4 with higher affinity for the receptor fraction than that of the wild type was identified. The increased affinity was attributed to the substitution of Glu(16) by Val(16). Although the substitution probably caused an increased entropic cost to the helix region, the linker around Val(16) is more flexible resulting in the increase of affinity for the receptor.

Comparison of Expression of Monomeric and Multimeric Adenoregulin Genes in Escherichia coli and Pichia pastorias

Adenoregulin is a 33 amino acid antibiotic peptide who belongs to dermaseptin family which is the first vertebrate family to show lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. Synthetic adenoregulin gene was cloned in 2, 4 and 6 tandem repeats and subcloned in pET32a and pET22b vectors. Recombinant plasmids were transformed into E. coli BL21(DE3), Fusion proteins of Trx-ADR1, Trx-ADR2 and Trx-ADR4 could be expressed after the hosts were induced by IPTG, but the expression level decreased dramatically with the number of tandem repeats increased. ADR1, ADR4 and ADR6 could not be expressed by E. coli without carrier proteins. But for Pichia pastoris GS115, ADR1 and ADR6 in the fermentation broth of the hosts could be detected by ELISA, and the bactericidal activities could also be observed.