Xiaolin Pei

Overexpression and characterization of a new organic solvent-tolerant esterase derived from soil metagenomic DNA

In this study, an esterase, designated EstC23, was isolated from a soil metagenomic library. The protein was amenable to overexpression in Escherichia coli under control of the T7 promoter, resulting in expression of the active, soluble protein that constituted 30% of the total cell protein content. This enzyme showed optimal activity at 40 °C and retained about 50% maximal activity at 5-10 °C. EstC23 showed remarkable stability in up to 50% (v/v) benzene and alkanes (high logP solvents). When incubated for 7 days in the presence of 50% benzene or alkanes, the enzyme maintained its 2-3 fold elevated activity. The purified enzyme also cleaved sterically hindered esters of tertiary alcohols. These results indicate that EstC23 has potential for use in industrial processes.

Prospecting metagenomic enzyme subfamily genes for dna family shuffling by a novel pcr-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.

Overexpression and characterization of a novel (S)-specific extended short-chain dehydrogenase/reductase from Candida parapsilosis

The gene encoding a putative protein from Candida parapsilosis CDC317 (CPE) was cloned and overexpressed in Escherichia coli. The protein was amenable to overexpression in E. coli and constituted 35% of the total cell protein content. The optimal activity was determined at pH 5.5 and 40°C with the substrate 4-chloro-3-oxobutanoate ethyl ester (COBE). The optical purity of the product was over 99% enantiomeric excess for the (S)-isomer, and the molar conversion yield of the product was 91.1%. The apparent k(m) value for COBE was 0.19±0.01mM, which is an order of magnitude lower than that of other enzymes in the literature.

Preparation, characterization and relative bioavailability of oral elemene o/w microemulsion

The objective was to develop an elemene oil/water (o/w) microemulsion and evaluate its characteristics and oral relative bioavailability in rats. Elemene was used as the oil phase and drug, polysorbate 80 as a surfactant along with ethanol, propylene glycol, and glycerol as the cosurfactants. The microemulsion was prepared by mixing method, or ultrasonication method in an ultrasonic bath. Its three-dimensional response surface diagram was drawn by Mathcad software. The microemulsion was characterized by visual observation, cross-polarized microscopy, size, zeta potential, acidity, viscosity, and surface tension measurement. The drug content and entrapment efficiency were determined by ultra fast liquid chromatography (UFLC) and liquid surface method. Blood was drawn from rats at different time points after oral administration of an elemene microemulsion or a commercial elemene emulsion for measurement of the drug in plasma by UFLC to establish the pharmacokinetic parameters and relative bioavailability. The elemene microemulsion as a clarified and isotropic system containing 1% elemene (w/v), 5% ethanol (v/v), 15% propylene glycol (v/v), 15% glycerol (v/v), and 5% polysorbate 80 (w/v), was characterized as (57.7 ± 2.8) nm in size, 0.485 ± 0.032 in polydispersity index, (3.2 ± 0.4) mv in zeta potential, (5.19 ± 0.08) in pH, 6 mpa·s in viscosity, (31.8 ± 0.3) mN·m−1 in surface tension, (8.273 ± 0.018) mg·mL−1 in content of β-elemene, and (99.81 ± 0.24)% in average entrapment efficiency. The area under the concentration-time curves from 0 h to 24 h (AUC0→24h) of the elemene microemulsion and commercial elemene emulsion were integrated to be 3.092 mg·h·L−1 and 1.896 mg·h·L−1 respectively, yielding a relative bioavailability of 163.1%. The present study demonstrates the elemene microemulsion as a new formulation with ease of preparation, high entrapment efficiency, excellent clarity, good stability, and improved bioavailability.

Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature

Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20°C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30°C. The total NHase activities reached to 263 and 155U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.

Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR

A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.

Identification and functional analysis of the activator gene involved in the biosynthesis of Co-type nitrile hydratase from Aurantimonas manganoxydans

Nitrile hydratase (NHase) has attracted considerable attention to synthesize valuable amides. Here, the gene cluster of Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229) was identified through Edman degradation and recombinant expression, including α-subunit, β-subunit, and activator. The activator gene contained 408 bases, which was essential for the biosynthesis of NHase1229 in Escherichia coli. The activator gene can be substituted by Co-type activator from Pseudonocardia thermophila JCM3095 and Pseudomonas putida 5B to assist in the functional expression of NHase1229, not by that of Fe-type NHase from Pseudomonas putida F1. Multi-sequence alignment showed that the activator contained conserved amino acid residues (Trp34, Trp53, Phe 85, Gln35, Glu39, and Glu90), but these residues had no significant effect on activator function. Histidine residues (His112, His120, and His134) at the C-terminus only partially participated in the activator function. Furthermore, the successive α-helices in domain TIGR03889 were identified to be critical for the activator function. The integration efficiency of cobalt into the active site was decreased with the interruption of α-helices. Therefore, Co-type and Fe-type activators have specific functions for the integration of different metal ions, and four conserved α-helices in Co-type activator affect the integration of cobalt. These knowledge is useful for understanding the biosynthesis of Co-type NHase.

Creation of Functionally Diverse Chimerical α-Glucosidase Enzymes by Swapping Homologous Gene Fragments Retrieved from Soil DNA

Abstractα-Glucosidase (XcG) from Xanthomonas campestris is an interesting enzyme due to its ability to catalyze transglycosylation reactions using maltose, rather than expensive nucleotide-activated sugars. In this study, two chimerical enzymes, XcG-A and XcG-B, were created by substituting the corresponding region of the XcG gene with gene fragments retrieved from metagenomic DNA samples. The enzymatic characterization results revealed that XcG-A exhibited a significant greater transglycosylation capacity for α-arbutin production and a significantly improved ability to produce α-arbutin-α-glycosides than XcG and XcG-B.

N-terminal engineering of overlapping genes in the nitrile hydratase gene cluster improved its activity

Nitrile hydratase which catalyzes the hydration of nitriles to the corresponding amides is operon-encoded. However, when heterologously expressed, genes in the same operon are usually not equally expressed, and the ratio needs to be fine-tuned. A gene cluster of three genes (corresponding to α-subunit, β-subunit and activator) encoding the nitrile hydratase was cloned from Aurantimonas manganoxydans ATCC BAA-1229 and expressed in Escherichia coli. However, difficulty was encountered in heterologous expression of the activator and the expression level of β-subunit was lower than that of α-subunit, which together resulted in low catalytic efficiency. To improve the expression of activator, a set of SKIK tags were fused to the N-terminus of the activator. To elevate the expression level of β-subunit, a silent mutation strategy was applied in the overlapping sequence with α-subunit around its translation initial region. Finally, the expression of β-subunit and activator were improved and the maximum activity of NHase1229 was doubled, reaching 160 U/mL towards 3-cyanopyridine. These results indicate that N-terminal engineering is an efficient strategy for optimizing the expression of multiple genes in operons.