Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (R)-Tebuconazole in High Optical Purity Mediated by an Epoxide Hydrolase from Rhodotorula paludigensis.

Tebuconazole is a chiral triazole fungicide used globally in agriculture as a racemic mixture, but its enantiomers exhibit significant enantioselective dissimilarities in bioactivity and environmental behaviors. The steric hindrance caused by the tert-butyl group makes it a great challenge to synthesize tebuconazole enantiomers. Here, we designed a simple chemoenzymatic approach for the asymmetric synthesis of (R)-tebuconazole, which includes the biocatalytic resolution of racemic epoxy-precursor (2-tert-butyl-2-[2-(4-chlorophenyl)ethyl] oxirane, rac-1a) by Escherichia coli/Rpeh whole cells expressed epoxide hydrolase from Rhodotorula paludigensis (RpEH), followed by a one-step chemocatalytic synthesis of (R)-tebuconazole. It was observed that (S)-1a was preferentially hydrolyzed by E. coli/Rpeh, whereas (R)-1a was retained with a specific activity of 103.8 U/g wet cells and a moderate enantiomeric ratio (E value) of 13.4, which was remarkably improved to 43.8 after optimizing the reaction conditions. Additionally, a gram-scale resolution of 200 mM rac-1a was performed using 150 mg/mL E. coli/Rpeh wet cells, resulting in the retention of (R)-1a in a 97.0% ees, a 42.5% yields, and a 40.5 g/L/d space-time yield. Subsequently, the synthesis of highly optical purity (R)-tebuconazole (>99% ee) was easily achieved through the chemocatalytic ring-opening of the epoxy-precursor (R)-1a with 1,2,4-triazole. To elucidate insight into the enantioselectivity, molecular docking simulations revealed that the unique L-shaped substrate-binding pocket of RpEH plays a crucial role in the enantioselective recognition of bulky 2,2-disubstituted oxirane 1a.

The role of cellular senescence-related genes in Asthma: Insights from bioinformatics and animal experiments.

BACKGROUND: Asthma is a heterogeneous chronic respiratory disease, affecting about 10% of the global population. Cellular senescence is a multifaceted phenomenon defined as the irreversible halt of the cell cycle, commonly referred to as the senescence-associated secretory phenotype. Recent studies suggest that cellular senescence may play a role in asthma. This study aims to dissect the role and biological mechanisms of CSRGs in asthma, enhancing our understanding of the progression of asthma. METHODS: The study utilized the GSE147878 dataset, employing methods like WGCNA, Differential analysis, Cibersort, GO, KEGG, unsupervised clustering, and GSVA to explore CSRGs functions and immune cell patterns in asthma. Machine learning identified key diagnostic genes, validated externally with the GSE165934 dataset and through qRT-PCR and WB experiments in animal models. RESULT: From the GSE147878 dataset, 24 CSRGs were identified, highlighting their role in immune and inflammatory processes in asthma. Differences in CD4 naive T cells and activated dendritic cells between asthma and control groups underscored CSRGs' role in immune regulation. Cluster analysis revealed two distinct asthma patient groups with unique immune microenvironments. Machine learning identified five genes, leading to a TF-miRNA-mRNA network and singling out RHOA and RBM39 as key diagnostic genes, which were experimentally validated. Finally, a nomogram was created based on these genes. CONCLUSION: This study, utilizing bioinformatics and animal experiments, identified RHOA and RBM39 as key diagnostic genes for asthma, providing new insights into the potential role and biological mechanisms of CSRGs in asthma.

Exploration of the Binding Mechanism of Cyclic Dinucleotide Analogs to Stimulating Factor Proteins and the Implications for Subsequent Analog Drug Design.

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiae and its purification and immobilization by one-step method.

Nicotinamide riboside kinase (NRK) plays an important role in the synthesis of ß -nicotinamide nucleotide (NMN). NMN is a key intermediate of NAD+ synthesis, and it actually contribute to the well-being of our health. In this study, gene mining technology was used to clone nicotinamide nucleoside kinase gene fragments from S. cerevisiae, and the ScNRK1 was achieved a high level of soluble expression in E. coli BL21. Then, the reScNRK1 was immobilized by metal affinity label to optimize the enzyme performance. The results showed that the enzyme activity in the fermentation broth was 14.75 IU/mL, and the specific enzyme activity after purification was 2252.59 IU/mg. After immobilization, the optimum temperature of the immobilized enzyme was increased by 10°C compared with the free enzyme, and the temperature stability was improved with little change in pH. Moreover, the activity of the immobilized enzyme remained above 80% after four cycles of immobilized reScNRK1, which makes the enzyme more advantageous in the enzymatic synthesis of NMN.

Gene cloning of a highly active phytase from Lactobacillus plantarum and further improving its catalytic activity and thermostability through protein engineering.

Phytase belongs to orthophosphate monoester hydrolase, which can catalyze the gradual hydrolysis of phytic acid to inositol phosphate. It can be added to animal feed to reduce the anti-nutritional factor of phytic acid in feed. The thermostability and specific activity of phytases are two key factors determining their potential applications. In this study, a highly active 233-aa phytase gene (LpPHY233) from Lactobacillus plantarum was cloned and expressed in Escherichia coli (E. coli), achieving 800 times higher activity than that expressed in L. plantarum. Next, the temperature characteristic and catalytic performance of LpPHY233 was improved by disulfide bond engineering and C-terminal truncation, respectively. Surprisingly, the specific activity of the C-terminal truncated mutant LpPHY200 was about 5.6 times higher than that of LpPHY233, and the optimal temperature for the mutant LpPHY233S58C/K61C introduced disulfide bond was 15 °C higher than that of LpPHY233. Moreover, these phytase mutants displayed excellent pH property and kinetic parameters, and have great application prospect in feed additives field. The molecular basis for its catalytic performance was preliminarily explained by in silico design methods. Our results provided a solid theoretical foundation for further molecular modification and industrial application of phytases.

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli.

Leucine dehydrogenase (LDH) is a NAD+-dependent oxidoreductase, which can selectively catalyze α-keto acids to obtain α-amino acids and their derivatives. It plays a key role in the biosynthesis of L-tert-leucine (L-Tle). As a non-naturally chiral amino acid, L-Tle can be used as an animal feed additive, nutrition fortifier, which is a perspective and important building block in the pharmaceutical, cosmetic, and food additive industry. In this study, four hypothetical leucine dehydrogenases were discovered by using genome mining technology, using the highly active leucine dehydrogenase LsLeuDH as a probe. These four leucine dehydrogenases were expressed in Escherichia coli BL21(DE3), respectively, and purified to homogeneity and characterized. Compared with the other enzymes, the specific activity of PfLeuDH also shows stronger advantage. In addition, the highly selective biosynthesis of L-Tle from trimethylpyruvic acid (TMP) was successfully carried out by whole-cell catalysis using engineered E. coli cells as biocatalyst, which can efficiently coexpress leucine dehydrogenase and formate dehydrogenase. One hundred-millimolar TMP was catalyzed for 25 h, and the yield and space-time yield of L-Tle reached 87.38% (e.e. >99.99%) and 10.90 g L-1 day-1. In short, this research has initially achieved the biosynthesis of L-Tle, laying a solid foundation for the realization of low-cost and large-scale biosynthesis of L-Tle.

Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli.

Glutamate decarboxylase (GAD) has the potential of converting L-glutamate to gamma-aminobutyric acid (GABA), which is an important non-proteinogenic amino acid that has a potential use as food additive or dietary supplement for its physiological functions. A novel pyridoxal 5'-phosphate (PLP)-dependent glutamate decarboxylase (LsGAD) was cloned from GRAS (generally recognized as safe) Lactobacillus senmaizukei by genome mining and efficiently expressed in Escherichia coli BL21. The LsGAD displayed excellent temperature property, pH property and kinetic parameters compared with the probe LbGAD and the other GADs. By increasing the copy number of the LsGAD encoding gene, the expression level of LsGAD and the biosynthesis yield of GABA were increased, which was near to 2 times of that was expressed in single copy. These results established a solid foundation for increasing the added value of L-glutamate and the biosynthesis of GABA.

High level and enantioselective production of L-phenylglycine from racemic mandelic acid by engineered Escherichia coli using response surface methodology.

L-Phenylglycine (L-PHG) is a member of unnatural amino acids, and becoming more and more important as intermediate for pharmaceuticals, food additives and agrochemicals. However, the existing synthetic methods for L-PHG mainly rely on toxic cyanide chemistry and multistep processes. To provide green, safe and high enantioselective alternatives, we envisaged cascade biocatalysis for the one-pot synthesis of L-PHG from racemic mandelic acid. A engineered E. coli strain was established to co-express mandelate racemase, D-mandelate dehydrogenase and L-leucine dehydrogenase and catalyze a 3-step reaction in one pot, enantioselectively transforming racemic mandelic acid to give L-PHG (e.e. >99 %). After the conditions for biosynthesis of L-PHG optimized by response surface methodology, the yield and space-time yield of L-PHG can reach 87.89 % and 79.70 g·L-1·d-1, which was obviously improved. The high-yielding and enantioselective synthetic methods use cheap and green reagents, and E. coli whole-cell catalysts, thus providing green and useful alternative methods for manufacturing L-PHG.

One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis.

Phenylglyoxylic acid (PGA) are key building blocks and widely used to synthesize pharmaceutical intermediates or food additives. However, the existing synthetic methods for PGA generally involve toxic cyanide and complex processes. To explore an alternative method for PGA biosynthesis, we envisaged cascade biocatalysis for the one-pot synthesis of PGA from racemic mandelic acid. A novel mandelate racemase named ArMR showing higher expression level (216.9 U·mL-1 fermentation liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of mandelate racemase, d-mandelate dehydrogenase and l-lactate dehydrogenase, and transform racemic mandelic acid to PGA. Among them, the recombinant E. coli TCD 04, engineered to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic acid (100 mM) to PGA with 98% conversion. Taken together, we provide a green approach for one-pot biosynthesis of PGA from racemic mandelic acid.

Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity.

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD+-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg-1, which was close to four times the activity of the probe. Meanwhile, the kcat/ Km value of LhDMDH was 28.80 S-1·mM-1, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.

Genome mining and motif truncation of glycoside hydrolase family 5 endo-ß-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

Two novel glycosyl hydrolase family 5 (GH5) ß-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9KM. The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant ß-mannanases potential additives for use in the food and feed industries.

Expression of a novel epoxide hydrolase of Aspergillus usamii E001 in Escherichia coli and its performance in resolution of racemic styrene oxide.

The full-length cDNA sequence of Aueh2, a gene encoding an epoxide hydrolase of Aspergillus usamii E001 (abbreviated to AuEH2), was amplified from the total RNA. Synchronously, the complete DNA sequence containing 5', 3' flanking regions, eight exons and seven introns was cloned from the genomic DNA. In addition, a cDNA fragment of Aueh2 encoding a 395-aa AuEH2 was expressed in Escherichia coli. The catalytic activity of recombinant AuEH2 (re-AuEH2) was 1.44 U/ml using racemic styrene oxide (SO) as the substrate. The purified re-AuEH2 displayed the maximum activity at pH 7.0 and 35 °C. It was highly stable at a pH range of 5.0-7.5, and at 40 °C or below. Its activity was not obviously influenced by ß-mercaptoethanol, EDTA and most of metal ions tested, but was inhibited by Hg(2+), Sn(2+), Cu(2+), Fe(3+) and Zn(2+). The K m and V max of re-AuEH2 were 5.90 mM and 20.1 U/mg towards (R)-SO, while 7.66 mM and 3.19 U/mg towards (S)-SO. Its enantiomeric ratio (E) for resolution of racemic SO was 24.2 at 10 °C. The experimental result of re-AuEH2 biasing towards (R)-SO was consistent with the analytical one by molecular docking (MD) simulation.

Cloning, expression of a feruloyl esterase from Aspergillus usamii E001 and its applicability in generating ferulic acid from wheat bran.

A cDNA gene (AufaeA), which encodes a mature polypeptide of the type-A feruloyl esterase from Aspergillus usamii E001 (abbreviated to AuFaeA), was cloned and heterologously expressed in Pichia pastoris GS115. One transformant, labeled as P. pastoris GSFaeA4-8, expressing the highest recombinant AuFaeA (reAuFaeA) activity of 10.76 U/ml was selected by the flask expression test. The expressed reAuFaeA was purified to homogeneity with an apparent molecular weight of 36.0 kDa by SDS-PAGE analysis, and characterized using the model substrate of methyl ferulate (MFA). The purified reAuFaeA was optimally active at pH 5.0 and 45 °C, and highly stable at pH 4.0-6.5 and 45 °C or below. Its activity was not significantly affected by metal ions tested and EDTA. The K m and V max of reAuFaeA towards MFA were 4.64 mM and 115.5 U/mg, respectively. High-performance liquid chromatography analysis showed that only 9.7 % of total alkali-extractable ferulic acid (FA) was released from destarched wheat bran by reAuFaeA alone. The released FA increased to 36.5 % when reAuFaeA was used together with a recombinant Aspergillus usamii GH family 11 xylanase A, indicating a synergistic interaction between them.

Fusing a carbohydrate-binding module into the Aspergillus usamii ß-mannanase to improve its thermostability and cellulose-binding capacity by in silico design.

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 ß-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion ß-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.

Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation.

BACKGROUND: Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement. RESULTS: The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn1157, was predicted by molecular dynamics (MD) simulation. An NhXyn1157-encoding gene, Nhxyn1157, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn1157 (re-NhXyn1157) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn1157 were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²âº and Ag⁺. CONCLUSION: This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn1157 a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.

Cloning and expression of a family 10 xylanase gene (Aoxyn10) from Aspergillus oryzae in Pichia pastoris.

A full-length cDNA sequence of Aoxyn10, a gene encoding a glycoside hydrolase (GH) family 10 xylanase from Aspergillus oryzae, was amplified from the total RNA by 3' and 5' rapid amplification of cDNA ends. The cDNA sequence is 1,689 bp, containing 5', 3' untranslated regions and a 1,422 bp open reading frame (ORF) that encodes a 21-aa signal peptide and a 452-aa mature peptide (designated AoXyn10). Multi-alignment revealed that AoXyn10 contains two regions: a catalytic domain (CD) and a family 1 carbohydrate-binding module (CBM1). The three-dimensional (3-D) structure of the CD was predicted by multiple template-based homology modeling. A 2,308-bp complete DNA sequence of Aoxyn10 was obtained from the genomic DNA by both pUCm-T vector-mediated and conventional PCRs, harboring 5', 3' flanking regulatory regions, five exons and four introns. Moreover, Aoxyn10 was extracellularly expressed in Pichia pastoris. One transformant labeled as P. pastoris GS/Xyn4-11 was selected, expressing the highest recombinant AoXyn10 (named reAoXyn10) activity of 45.0 U/ml. SDS-PAGE analysis revealed that reAoXyn10, a glycoprotein with an apparent molecular weight (M.W.) of about 56.0 kDa, was secreted into the cultured medium. The purified reAoXyn10 displayed the maximum activity at pH 5.5 and 60°C. It was stable at a pH range of 4.0-7.0, and at 50°C or below. Its activity was not affected by an array of metal ions or EDTA, but was inhibited by Mn(2+) and Ba(2+). The Km and Vmax of reAoXyn10 were 1.7 mg/ml and 817 µmol/min/mg, respectively.

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme.

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K(M) vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K(M)-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5-8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn(2+), which caused a strong inhibition. The K(m) and V(max), towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-ß-1,4-D-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris.

hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His+, Mut+) with multiple hly copies. One transformant resistant to 4.0 mg/ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol.