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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.

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.

Two thermophilic β-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.

The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A-base overhang. The other vector pYEVB, with the same features for blunt-end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X-gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full-length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by SalI and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time-consuming conventional cloning procedures.