Xiaopu Yin

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.

A novel and efficient method for the immobilization of thermolysin using sodium chloride salting-in and consecutive microwave irradiation.

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency.

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.

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.

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.

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.

Assessing the stereoselectivity of carbonyl reductases toward the reduction of OPBE and docking analysis

The asymmetric reduction of prochiral carbonyl compounds by NAD(P)H‐dependent carbonyl reductases represents a powerful method for the production of optically active alcohols. The stereoselectivity of a series of carbonyl reductases were evaluated toward the reduction of ethyl 2‐oxo‐4‐phenylbutyrate (OPBE). A majority of reductases produced the ethyl (R)‐2‐hydroxy‐4‐phenylbutyrate ((R)‐HPBE) with low to excellent enantiomeric excess (e.e.), whereas about 30% reductases catalyzed OPBE to form (S)‐HPBE. Among them, the carbonyl reductase from Saccharomyces cerevisiae (SeCR) and short‐chain dehydrogenase from Gluconobacter oxydans (GoKR) exhibited 100% e.e., yielding the corresponding (R) and (S)‐HPBE, respectively. However, the SeCR showed relative higher activity (29.0 U/mg) and affinity (Km of 0.22 mM) than those of GoKR. Docking analysis found that the interaction of OPBE with enzyme‐NADPH complex determined the NADPH‐provided hydrogen transfer and the configuration of reductive product. These results indicated that the three‐dimensional (3D) structure of enzymes controlled the stereoselectivity of the reductive product based on the geometry of the substrate and cofactor.