Supplementation with Exogenous Catalase from Penicillium notatum in the Diet Ameliorates Lipopolysaccharide-Induced Intestinal Oxidative Damage through Affecting Intestinal Antioxidant Capacity and Microbiota in Weaned Pigs.

The present study aimed to explore the protective effects of exogenous catalase (CAT) from microorganisms against lipopolysaccharide (LPS)-induced intestinal injury and its molecular mechanism in weaned pigs. Fifty-four weaned pigs (21 days of age) were randomly allocated to CON, LPS, and LPS+CAT groups. The pigs in CON and LPS groups were fed a basal diet, whereas the pigs in LPS+CAT group fed the basal diet with 2,000 mg/kg CAT supplementation for 35 days. On day 36, six pigs were selected from each group, and LPS and LPS+CAT groups were administered with LPS (50 µg/kg body weight). Meanwhile, CON group was injected with an equivalent amount of sterile saline. Results showed that LPS administration damaged intestinal mucosa morphology and barrier. However, CAT supplementation alleviated the deleterious effects caused by LPS challenge through enhancing intestinal antioxidant capacity which was benefited to decrease proinflammatory cytokines concentrations and suppress enterocyte apoptosis. Besides, LPS-induced gut microbiota dysbiosis was significantly shifted by CAT through decreasing mainly Streptococcus and Escherichia-Shigella. Our study suggested that dietary supplemented with 2,000 mg/kg catalase was conducive to improve intestinal development and protect against LPS-induced intestinal mucosa injury via enhancing intestinal antioxidant capacity and altering microbiota composition in weaned pigs. IMPORTANCE Exogenous CAT derived from microorganisms has been widely used in food, medicine, and other industries. Recent study also found that exogenous CAT supplementation could improve growth performance and antioxidant capacity of weaned pigs. However, it is still unknown that whether dietary exogenous CAT supplementation can provide a defense against the oxidative stress-induced intestinal damage in weaned pigs. Our current study suggested that dietary supplemented with 2,000 mg/kg CAT was conducive to improve intestinal development and protect against LPS-induced intestinal mucosa injury via enhancing intestinal antioxidant capacity and altering microbiota composition in weaned pigs. Moreover, this study will also assist in developing of CAT produced by microorganisms to attenuate various oxidative stress-induced injury or diseases.

Rational design of a highly efficient catalytic system for the production of PAPS from ATP and its application in the synthesis of chondroitin sulfate.

The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of adenosine triphosphate (ATP) (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate.

Tyrosinase from Penicillium chrysogenum: Characterization and application in phenol removal from aqueous solution.

The tyrosinase of Penicillium chrysogenum strain AUMC 14100 Accession No. MN219732 was purified to homogeneity and chemically modified by N-ethylmaleimide (NEM) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride, DC). The inactivation of the purified enzyme obeyed pseudo-first-order reaction kinetics in the presence of NEM and DC (1-5 mM). The rate constants of the enzyme inactivation by NEM and DC were calculated to be 0.083 mol/min and 0.0013 mol/min, respectively. The recovery of enzyme activity by the protective effect of substrate indicates a non-specific modification of the active center. The order of tyrosinase inactivation kinetics and the substrate protection revealed the essentiality of sulfhydryl and lysyl residues in the enzyme active site and its role in the enzyme catalysis. The immobilized tyrosinase on alginate showed a gradual increase in residual activity over the immobilization time until the fourth hour. The desorptivity of tyrosinase was gradually raised with higher sodium dodecyl sulfate (SDS) concentrations. The immobilized enzyme retained about 70% of its original activity after 8 repeated cycles. Thus, immobilized tyrosinase of Penicillium chrysogenum removed 75% of phenol after 8 cycles and thus seems likely to be a good candidate for phenol removal in aqueous solution.

Detection, Purification and Elucidation of Chemical Structure and Antiproliferative Activity of Taxol Produced by Penicillium chrysogenum.

Penicillium chrysogenum has been reported as a potent taxol producer based on quantitative analysis by TLC and HPLC. The biosynthetic potency of taxol has been validated from PCR detection of rate-limiting genes of taxol synthesis such as taxadienesynthase and 10-de-acetylbaccatin III-O-acetyltransferase (DBAT), which catalyzes the immediate diterpenoid precursor of the taxol substance, as detected by PCR. Taxol production by P. chrysogenum was assessed by growing the fungus on different media. Potato dextrose broth (PDB) was shown to be the best medium for obtaining the higher amount of taxol (170 µg/L). A stepwise optimization of culture conditions necessary for production of higher amounts of taxol was investigated. The substance taxol was produced optimally after 18 d of incubation at 30 °C in PDB adjusted initially at pH 8.0 with shaking (120 rpm) (250 µg/L). The P. chrysogenum taxol was purified successfully by HPLC. Instrumental analyzes such as Fourier transform infrared spectroscopy (FTIR), ultraviolet (UV) spectroscopy, 1HNMR and 13C NMR approved the structural formula of taxol (C47H51NO14), as constructed by ChemDraw. The P. chrysogenum taxol showed promising anticancer activity.

The same but different: multiple functions of the fungal flavin dependent monooxygenase SorD from Penicillium chrysogenum.

Sorbicillinoids are a large family of fungal secondary metabolites with a diverse range of structures and numerous bioactivites, some of which have pharmaceutical potential. The flavin-dependent monooxygenase SorD from Penicillium chrysogenum (PcSorD) utilizes sorbicillinol to catalyze a broad scope of reactions: formation of oxosorbicillinol and epoxysorbicillinol; intermolecular Diels-Alder and Michael-addition dimerization reactions; and dimerization of a sorbicillinol derivative with oxosorbicillinol. PcSorD shares only 18.3% sequence identity with SorD from Trichoderma reesei (TrSorD) and yet unexpectedly catalyzes many of the same reactions, however, the formation of oxosorbicillinol and bisvertinolone by PcSorD extends the range of reactions catalyzed by a single enzyme. Phylogenetic analysis indicates that PcSorD and TrSorD bind the flavin cofactor covalently but via different residues and point mutations confirm these residues are essential for activity.

Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Identification and characterization of ferulic acid esterase from Penicillium chrysogenum 31B: de-esterification of ferulic acid decorated with l-arabinofuranoses and d-galactopyranoses in sugar beet pectin.

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg2+, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-ß-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-ß-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both ß-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.

Cooperation of hydrolysis modes among xylanases reveals the mechanism of hemicellulose hydrolysis by Penicillium chrysogenum P33.

BACKGROUND: Xylanases randomly cleave the internal ß-1,4-glycosidic bonds in the xylan backbone and are grouped into different families in the carbohydrate-active enzyme (CAZy) database. Although multiple xylanases are detected in single strains of many filamentous fungi, no study has been reported on the composition, synergistic effect, and mode of action in a complete set of xylanases secreted by the same microorganism. RESULTS: All three xylanases secreted by Penicillium chrysogenum P33 were expressed and characterized. The enzymes Xyl1 and Xyl3 belong to the GH10 family and Xyl3 contains a CBM1 domain at its C-terminal, whereas Xyl2 belongs to the GH11 family. The optimal temperature/pH values were 35 °C/6.0, 50 °C/5.0 and 55 °C/6.0 for Xyl1, Xyl2, and Xyl3, respectively. The three xylanases exhibited synergistic effects, with the maximum synergy observed between Xyl3 and Xyl2, which are from different families. The synergy between xylanases could also improve the hydrolysis of cellulase (C), with the maximum amount of reducing sugars (5.68 mg/mL) observed using the combination of C + Xyl2 + Xyl3. Although the enzymatic activity of Xyl1 toward xylan was low, it was shown to be capable of hydrolyzing xylooligosaccharides into xylose. Xyl2 was shown to hydrolyze xylan to long-chain xylooligosaccharides, whereas Xyl3 hydrolyzed xylan to xylooligosaccharides with a lower degree of polymerization. CONCLUSIONS: Synergistic effect exists among different xylanases, and it was higher between xylanases from different families. The cooperation of hydrolysis modes comprised the primary mechanism for the observed synergy between different xylanases. This study demonstrated, for the first time, that the hydrolysates of GH11 xylanases can be further hydrolyzed by GH10 xylanases, but not vice versa.

Bacterial MbtH-like Proteins Stimulate Nonribosomal Peptide Synthetase-Derived Secondary Metabolism in Filamentous Fungi.

Filamentous fungi are known producers of bioactive natural products, low molecular weight molecules that arise from secondary metabolism. MbtH-like proteins (MLPs) are small (∼10 kDa) proteins, which associate noncovalently with adenylation domains of some bacterial nonribosomal peptide synthetases (NRPS). MLPs promote the folding, stability, and activity of NRPS enzymes. MLPs are highly conserved among a wide range of bacteria; however, they are absent from all fungal species sequenced to date. We analyzed the interaction potential of bacterial MLPs with eukaryotic NRPS enzymes first using crystal structures, with results suggesting a conservation of the interaction surface. Subsequently, we transformed five MLPs into Penicillium chrysogenum strains and analyzed changes in NRPS-derived metabolite profiles. Three of the five transformed MLPs increased the rate of nonribosomal peptide formation and elevated the concentrations of intermediate and final products of the penicillin, roquefortine, chrysogine, and fungisporin biosynthetic pathways. Our results suggest that even though MLPs are not found in the fungal domain of life, they can be used in fungal hosts as a tool for natural product discovery and biotechnological production.

Structural and functional characterisation of xylanase purified from Penicillium chrysogenum produced in response to raw agricultural waste.

Commercial interest in plant cell wall degrading enzymes (PCWDE) is motivated by their potential for energy or bioproduct generation that reduced dependency on non-renewable (fossil-derived) feedstock. Therefore, underlying work analysed the Penicillium chrysogenum isolate for PCWDE production by employing different biomass as a carbon source. Among the produced enzymes, three xylanase isoforms were observed in the culture filtrate containing sugarcane bagasse. Xylanase (PcX1) presenting 35 kDa molecular mass was purified by gel filtration and anion exchange chromatography. Unfolding was probed and analysed using fluorescence, circular dichroism and enzyme assay methods. Secondary structure contents were estimated by circular dichroism 45% α-helix and 10% ß-sheet, consistent with the 3D structure predicted by homology. PcX1 optimally active at pH 5.0 and 30 °C, presenting t1/2 19 h at 30 °C and 6 h at 40 °C. Thermodynamic parameters/melting temperature 51.4 °C confirmed the PcX1 stability at pH 5.0. PcX1 have a higher affinity for oat spelt xylan, KM 1.2 mg·mL-1, in comparison to birchwood xylan KM 29.86 mg·mL-1, activity was inhibited by Cu+2 and activated by Zn+2. PcX1 exhibited significant tolerance for vanillin, trans-ferulic acid, ρ-coumaric acid, syringaldehyde and 4-hydroxybenzoic acid, activity slightly inhibited (17%) by gallic and tannic acid.

Trehalose mediated stabilisation of cellobiase aggregates from the filamentous fungus Penicillium chrysogenum.

Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 µg of sugar/µg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins.

Effect of glucose depletion during the synthesis of galactooligosaccharides using a trienzymatic system.

In this study the effect of glucose depletion using glucose oxidase and catalase, simultaneously to the synthesis of prebiotic galactooligosaccharides (GOS) by ß-galactosidase was studied. Considering total GOS yield, a strong dependency on the source of ß-galactosidase was found. Using an Aspergillus oryzae lactase, a small increase in GOS yield (from 50.0 ± 1.3 g/L to 54.1 ± 1.9 g/L) was noted. Due to the decreased rate of GOS disaccharide formation by the Kluyveromyces lactis enzyme, the total GOS yield was diminished (from 47.4 ± 0.1 g/L to 30.5 ± 1.7 g/L). However, for both enzymes, the synthesis of GOS tri- and higher oligosaccharides increased. Additionally, the total sugar content, and thus caloric value, of the final product was reduced, also resulting in a more purified GOS mixture.

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents.

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55 °C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72 h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.

Production, purification, and biochemical characterization of serine alkaline protease from Penicillium chrysogenium strain X5 used as excellent bio-additive for textile processing.

A new ascomycete fungus X5, a hyperproducer (9000 U/mL) of a serine alkaline protease (SAPTEX) was identified as Penicillium chrysogenum. The experimental purification protocol comprises three steps: heat treatment (10 min at 80 °C) followed by an ammonium sulfate precipitation (30-50%)-dialysis, and a UNO Q-12 anion exchange chromatography using the FPLC system. The chemical characterizations performed include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 43,074.11 Da. The 25 residue NH2-terminal sequence of the enzyme showed high homology with Penicillium proteases. The optimum pH and temperature values for protease activity were pH 10 and 80 °C, respectively. Compared to other proteases (SPTC; Flavourzyme® 500 L; Proteinase, type XXIII; Proteinase K; and Alcalase® 2.4 L), SAPTEX has the highest catalytic efficacy, hydrolysis degree, and a powerful stability toward some commercial detergents. According to morphological, physico-chemical [scanning electron microscopy (SEM), energy dispersive X-Ray analysis (EDX), and FTIR-Fourier transform infrared spectroscopy], and mechanical evaluation, SAPTEX has no destructive impact on fibers after the enzyme treatment and a very slight effect on textile support. Obtained results suggested that SAPTEX may be considered as a potential candidate as a protein stain removal product for textile supports.

The N-terminal amphipathic helix of Pex11p self-interacts to induce membrane remodelling during peroxisome fission.

Pex11p plays a crucial role in peroxisome fission. Previously, it was shown that a conserved N-terminal amphipathic helix in Pex11p, termed Pex11-Amph, was necessary for peroxisomal fission in vivo while in vitro studies revealed that this region alone was sufficient to bring about tubulation of liposomes with a lipid consistency resembling the peroxisomal membrane. However, molecular details of how Pex11-Amph remodels the peroxisomal membrane remain unknown. Here we have combined in silico, in vitro and in vivo approaches to gain insights into the molecular mechanisms underlying Pex11-Amph activity. Using molecular dynamics simulations, we observe that Pex11-Amph peptides form linear aggregates on a model membrane. Furthermore, we identify mutations that disrupted this aggregation in silico, which also abolished the peptide's ability to remodel liposomes in vitro, establishing that Pex11p oligomerisation plays a direct role in membrane remodelling. In vivo studies revealed that these mutations resulted in a strong reduction in Pex11 protein levels, indicating that these residues are important for Pex11p function. Taken together, our data demonstrate the power of combining in silico techniques with experimental approaches to investigate the molecular mechanisms underlying Pex11p-dependent membrane remodelling.

Crystal structure of exo-rhamnogalacturonan lyase from Penicillium chrysogenum as a member of polysaccharide lyase family 26.

Exo-rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate-binding orientation and substrate specificity, PcRGLX is an exo-type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX-complex structures with reaction products indicate that the active site possesses an L-shaped cleft that can accommodate galactosyl side chains, suggesting side-chain-bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design.

Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of ß-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme.

A novel bifunctional acetyl xylan esterase/arabinofuranosidase from Penicillium chrysogenum P33 enhances enzymatic hydrolysis of lignocellulose.

BACKGROUND: Xylan, the major constituent of hemicellulose, is composed of ß-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes. RESULTS: Here, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6-9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-L-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency. CONCLUSIONS: The novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-L-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.

Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations and glucosylates from only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically 15N-labeled interleukin 8 (IL-8) C-terminal (34-72) glycopeptides bearing a Man9GlcNAc2 (M9) oligosaccharide. Chemical shift perturbation mapping NMR experiments suggested that Phe65 of the glycopeptide specifically interacts with UGGT. To analyze this interaction, we constructed a glycopeptide library by varying Phe65 with 10 other natural amino acids, via parallel native chemical ligation between a glycopeptide-α-thioester and a peptide library consisting of 11 peptides. UGGT assay against the glycopeptide library revealed that, although less hydrophobic glycopeptides could be used as substrates for UGGT, hydrophobic glycopeptides are preferred.

Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus.

Monacolin J is a key precursor for the synthesis of simvastatin (Zocor), an important drug for treating hypercholesterolemia. Industrially, monacolin J is manufactured through alkaline hydrolysis of lovastatin, a fungal polyketide produced by Aspergillus terreus. Multistep chemical processes for the conversion of lovastatin to simvastatin are laborious, cost expensive and environmentally unfriendly. A biocatalysis process for monacolin J conversion to simvastatin has been developed. However, direct bioproduction of monacolin J has not yet been achieved. Here, we identified a lovastatin hydrolase from Penicillium chrysogenum, which displays a 232-fold higher catalytic efficiency for the in vitro hydrolysis of lovastatin compared to a previously patented hydrolase, but no activity for simvastatin. Furthermore, we showed that an industrial A. terreus strain heterologously expressing this lovastatin hydrolase can produce monacolin J through single-step fermentation with high efficiency, approximately 95% of the biosynthesized lovastatin was hydrolyzed to monacolin J. Our results demonstrate a simple and green technical route for the production of monacolin J, which makes complete bioproduction of the cholesterol-lowering drug simvastatin feasible and promising.