Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.
Glucose Oxidase/chemistry* ; Enzyme Stability ; Aspergillus/genetics* ; Pichia/metabolism* ; Temperature ; Catalysis ; Fungal Proteins/metabolism* ; Hot Temperature

To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.
Single-Chain Antibodies/immunology* ; Humans ; Tetraspanin 30/immunology* ; Recombinant Proteins/immunology* ; Pichia/genetics* ; Saccharomycetales/metabolism*

Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
L-Lactate Dehydrogenase ; genetics ; Lactobacillus casei ; enzymology ; genetics ; Phenylpyruvic Acids ; metabolism ; Pichia ; genetics ; Recombinant Proteins ; genetics ; metabolism

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.
Carboxylic Ester Hydrolases ; metabolism ; Catalysis ; Molecular Weight ; Pichia ; genetics ; metabolism ; Polymerization ; Protein Engineering ; Substrate Specificity

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Escherichia coli ; Green Fluorescent Proteins ; Hydrophobic and Hydrophilic Interactions ; Peptides ; Pichia ; Recombinant Fusion Proteins ; metabolism

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.
Adaptation, Physiological ; Butanols ; metabolism ; Environment ; Fermentation ; Pichia ; cytology ; metabolism

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
Acrosome ; enzymology ; Animals ; Blotting, Western ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; Female ; Fertilization ; physiology ; Free Radical Scavengers ; metabolism ; Humans ; Hyaluronic Acid ; metabolism ; Male ; Muramidase ; analysis ; physiology ; Pichia ; Plasmids ; metabolism ; Recombinant Proteins ; analysis ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; enzymology ; Testis