High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

Efficient whole-cell biocatalyst for 2,3-butanediol/acetoin production with NADH/NAD+ regeneration system in engineered Serratia marcescens.

Serratia marcescens is reported to possess the potential for industrial 2,3-butanediol or acetoin production by fermentation. But 2,3-butanediol or acetoin are always co-produced and this may make purification process difficult. So biocatalytic technologies may be the appropriate production methods. In this study, we developed an auto-inducing expression system based on pET system and swr quorum sensing system by using S. marcescens. By using this system, S. marcescens could be engineered as whole-cell biocatalyst to obtain 2,3-butanediol or acetoin. In order to convert diacetyl to 2,3-butanediol, formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were co-expressed to construct a NADH regeneration system. This whole-cell biocatalyst could efficiently produced 53.6 g/L 2,3-butanediol with a productivity of 3.35 g/Lh. Next, in order to convert 2,3-butanediol to acetoin, NADH oxidase and 2,3-butanediol dehydrogenase were co-expressed to construct a NAD+ regeneration system. This whole-cell biocatalyst could efficiently produced 59 g/L acetoin with a productivity of 2.95 g/Lh. This work indicated this auto-inducing system was a powerful tool to construct whole-cell biocatalyst in S. marcescens in 2,3-butanediol and acetoin production.

High-level expression of two thermophilic ß-mannanases in Yarrowialipolytica.

Two thermophilic ß-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.

High-level expression of l-glutamate oxidase in Pichia pastoris using multi-copy expression strains and high cell density cultivation.

l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at µG = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at µM = 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.