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Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

Chitin is a linear homopolymer of acetylated β-(1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) from Aspergillus oryzae NKY2017 obtained from Hu’bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed in Pichia pastoris and subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels of CCHA expression in multi-copy strains, with strain CCHA4 chosen for high-density fermentation and enzyme-activity experiments. High-density fermentation achieved a CCHA yield of 22,500 U/mL, and temperature and pH optimization resulted in the highest CCHA activity at 40°C and 4.0, respectively. We used this enzyme for a large-scale preparation of oligosaccharides: 4 g enzyme could convert 150 kg chitosan into oligosaccharides in 24 h at 40°C. These results demonstrated abundant CCHA expression in P. pastoris and suggested the efficacy of CCHA for use in industrial applications.

L-amino acid oxidase (AAO) was reported to be capable of converting L-glutamic acid to α-aketoglutaric acid (α-KG). The sequence of AAO from Kitasatospora cheerisanensis was synthesized based on Pichia pastoris codon-usage preferences. AAO gene was cloned into plasmid pPICZα which was transformed into P. pastoris. Next, multi-copy expression plasmids were constructed by using plasmid pHBM905BDM. High-density fermentation was performed and the recombinant enzyme was characterized. The conversion conditions were optimized. By using Escherichia coli expression system, no soluble or active AAO was obtained from two strains after fermentation and induction. We can’t obtain high-level expression of recombinant strains by using plasmid pPICZα. Therefore, we constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PAAO1, PAAO2, PAAO3, PAAO4, and PAAO5, respectively. The following results showed that expression of AAO in multicopy strains increased as designed and strain PAAO5 was chosen for high-density fermentation and enzyme activity experiments. After high-density fermentation, we achieved an AAO-expression yield of 120.8 U/mL. After temperature and pH optimization, the highest AAO activity was observed at a temperature and pH of 20°C and 6, respectively. After optimization of the conversion conditions, the average production rate of L-glutamic acid to α-KG was 3.46 g/L/h and the highest α-KG titer (103 g/L) was converted from 120 g/L L-glutamic acid. In this study, AAO was abundantly expressed by using P. pastoris expression system. The following experiments indicated that AAO is suitable for use in industrial applications.

Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

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.

To seek a feasible technique for processing waste printed circuit boards (PCBs), pretreatment of PCBs by table separation and further bioleached by moderate thermophiles in a stirred tank reactor were investigated. The shaking table separation, conducted after grinding and sieving of PCBs, produced two fractions: metal-rich parts (RPCBs), which is more suitable for pyrometallurgy process than untreated PCBs, and metal-poor parts (PPCBs) with only 8.83% metals was then bioleached by a mixed culture of moderate thermophiles effectively. After adaptation, the mixed culture could tolerate 80 g/L PPCBs. The bioleaching results showed that metals recovery was 85.23% Zn, 76.59% Cu and 70.16% Al in only 7 days. Trace Pb and Sn were detected in the leachate because of precipitating. The microorganism community structure was analyzed by amplified ribosomal DNA restriction analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus. Furthermore, uncultured Thermoplasmatales archaeon was also detected in the leaching system. It was also shown that moderate thermophiles revealed best bioleaching ability when compared with mesophiles and the mixture of mesophiles and moderate thermophiles. Finally, we designed a two-stage process model according to the present study to achieve semi-industrial waste PCBs recycling and economic feasibility analysis indicated that the process was profitable.

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.