Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation activities toward chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the released reducing sugars the amount of (GlcNAc)2 was increased by 149.5%, whereas the amount of GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1, rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action.Keywords: chitin; Chitinase; Coprinopsis cinerea; hydrolysis; transglycosylation;

A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)3–5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)3–5; when the substrate was (GlcNAc)6–7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+β domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities.Keywords: chitin; chitin oligosaccharide; chitinase; Coprinopsis cinerea; fungal cell walls;

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography–mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.

Puerarin-7-O-fructoside was transformed by Trichoderma harzianum CGMCC 1523 into 3′-hydroxypuerarin-7-O-fructoside; this was identified by MS and NMR. However, puerarin-7-O-glucoside was not directly hydroxylated but hydrolyzed back into puerarin, which was transformed into 3′-hydroxypuerarin by the same fungi. Comparative analysis of free radical scavenging activity of DPPH showed that the free radical scavenging activity of puerarin-7-O-glucoside was reduced to approximately 1/2 of that of puerarin, while the free radical scavenging activity of puerarin-7-O-fructoside was increased to approximately 1.5 times of that of puerarin. The free radical scavenging activity of 3′-hydroxypuerarin-7-O-fructoside was further increased by 2.2 times of that of puerarin-7-O-fructoside, which was close to that of 3′-hydroxypuerarin.

A yeast identified as Rhodotorula mucilaginosa strain IM-2 was able to degrade acetamiprid (AAP) and thiacloprid (THI) in sucrose mineral salt medium with half-lives of 3.7 and 14.8 days, respectively, while it did not degrade imidacloprid and imidaclothiz. Identification of metabolites indicated that R. mucilaginosa IM-2 selectively converted AAP and THI by hydrolysis of AAP to form an intermediate metabolite IM 1-3 and hydrolysis of THI to form an amide derivative, respectively. Metabolite IM 1-3 had no insecticidal activity, while the THI amide showed considerable insecticidal activity but was 15.6 and 38.6 times lower than the parent THI following oral ingestion and a contact test against the horsebean aphid Aphis craccivora, respectively. The inoculated R. mucilaginosa IM-2 displayed biodegradability of AAP and THI in clay soils.

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 ± 31.9 µM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 ± 48.0 µM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, 13C NMR, 1H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 ± 5.4 µM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 ± 27.7 µM puerarin-7-O-glucoside and 187.8 ± 29.5 µM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 ± 24.0 µM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product’s composition.

A nicotinate dehydrogenase (NaDH) gene cluster was cloned from Comamonas testosteroni JA1. The enzyme, termed NaDHJA1, is composed of 21, 82, and 46 kDa subunits, respectivley containing [2Fe2S], Mo(V) and cytochrome c domains. The recombinant NaDHJA1 can catalyze the hydroxylation of nicotinate and 3-cyanopyridine. NaDHJA1 protein exhibits 52.8% identity to the amino acid sequence of NaDHKT2440 from P. putida KT2440. Sequence alignment analysis showed that the [2Fe2S] domain in NaDHJA1 had a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, while the same domain in NaDHKT2440 had only a type II [2Fe-2S] motif. NaDHKT2440 had an additional hypoxanthine dehydrogenase motif that NaDHJA1 does not have. When the small unit of NaDHJA1 was replaced by the small subunit from NaDHKT2440, the hybrid protein was able to catalyze the hydroxylation of nicotinate, but lost the ability to catalyze hydroxylation of 3-cyanopyridine. In contrast, after replacement of the small subunit of NaDHKT2440 with the small subunit from NaDHJA1, the resulting hybrid protein NaDHJAS+KTL acquired the ability to hydroxylate 3-cyanopyridine. The subunits swap results indicate the [2Fe2S] motif determines the 3-cyanopyridine hydroxylation ability, which is evidently different from the previous belief that the Mo motif determines substrate specificity.

6-Hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.

Chloropyridinyl neonicotinoid insecticides play a major role in crop protection and flea control on cats and dogs. Imidacloprid, thiacloprid and acetamiprid have in common the 6-chloro-3-pyridinylmethyl group but differ in the nitroguanidine or cyanoamidine substituent on an acyclic or cyclic moiety. Our previous study found that Stenotrophomonas maltophilia CGMCC 1.1788 could hydroxylate imidacloprid to 5-hydroxy imidacloprid, and 5-hydroxy imidacloprid was easily converted to 10–19 times higher insecticidal olefin imidacloprid against aphid or whitefly. Acetamiprid could be transformed by S. maltophilia to form N-demethylation product(IM 2-1). In this paper, we examined S. maltophilia CGMCC 1.1788’s ability of transformation of thiacloprid. S. maltophilia CGMCC 1.1788 can hydroxylate thiacloprid to 4-hydroxy thiacloprid characterized by HPLC-MS/MS and NMR analysis, however 4-hydroxy thiacloprid could not be converted to olefin thiacloprid under acid conditions like imidacloprid, whereas oxidized and decyonated simultaneously to form 4-ketone thiacloprid imine in alkaline solution. Bioassays indicated that 4-hydroxy thiacloprid had 156 times lower insecticidal activity than thiacloprid, and the ketone-imine derivative almost had no toxicity towards aphid. Though both imidacloprid and thiacloprid are hydroxylated by S. maltophilia CGMCC 1.1788 at the same carbon atom position, however the structural difference between in imidacloprid and thiacloprid, originate the entire discrepancy in bioefficacy of metabolite and its further degrading pathway. These results explain that why thiacloprid is classified as not relevant grade for soil and seed applications, whereas imidacloprid is recommended and acetamiprid is limited.

Microbacterium oxydans strain NJ 6 isolated from soil samples converted puerarin into two novel compounds, puerarin-7-O-glucoside and puerarin-7-O-isomaltoside, via an unreported O-glycosylation of the phenolic hydroxyl group at the 7-position of puerarin. Sucrose, maltotriose, and maltose could be used as glucosyl donors for glycosylation of puerarin, but uridine-diphosphate glucose, glucose, fructose, lactose, cyclodextrin, and starch could not. Regardless of the position of B-ring in the (iso)flavonoids core structure, the glycosylation of the phenolic hydroxyl group at the 7-position of (iso)flavonoids was governed by the presence or absence of a glucosyl residue at 8-C. The apparent solubility of puerarin-7-O-glucoside and puerarin-7-O-isomaltoside was approximately 18 and 100 times that of natural puerarin, respectively. Like parent puerarin, puerarin-7-O-glucoside maintained its physiological ability to relax the contractions of isolated rat thoracic aortic rings in vitro induced by phenylephrine. However, puerarin-7-O-glucoside was able to maintain higher plasma concentrations and have a longer mean residence time in the blood than the parent puerarin.

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni2+-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml−1 medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn2+, Ca2+, Fe2+, Mg2+ and Fe3+ ions stimulated the pectate lyase activity, but Cu2+ and Zn2+ inhibited it. The recombinant PelA had a Vmax of 77 μmol min−1 mg−1 and an apparent Km of 0.50 mg ml−1 for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S.maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.

•Providing a gentle and convenient way to explore the architecture of stipe cell walls.•Cell wall architecture varies with different stipe regions.•Modification of wall architecture is related to the loss of elongation in stipe base.A large amount of granular protrusions overlie the outer cell wall surfaces in both elongating and non-elongating stipe regions but overlie the inner cell wall surfaces only in non-elongating stipe regions. Removal of granular protrusions using alkali, amorphous materials overlying on both the inner and outer cell wall surfaces were explored in the non-elongating stipe regions. β-1,3-Glucanase treatment not only removed above those granular protrusions and underlying amorphous materials on the wall surfaces but also removed wall matrices embedding chitin microfibrils on the cell walls of most stipe regions, except for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained. The chitin microfibrils were closely and transversely arranged on both the inner and outer cell wall surfaces in the elongating apical stipe region, whereas they were loosely and transversely arranged on the inner cell wall surfaces and further became sparser and even randomly arranged on the outer cell wall surface in the non-elongating stipe regions. We propose that the surface deposition of granular protrusions and amorphous materials and the change of microfibril architecture and wall matrices may cause loss of wall plasticity and cessation of stipe elongation.

•ENG16A highly expresses during the mycelium stage.•Adjacent β-1,4-bonds favours ENG16A hydrolysis of β-1,3-glycosidic bonds.•Adjacent β-1,6-bonds hinders ENG16A hydrolysis of β-1,3-glycosidic bonds.•An endo-β-1,3(4)-glucanase corresponds to nutrient degradation.A gene coding endo-β-1,3(4)-glucanase (ENG16A) was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris. ENG16A only acts on substrates containing β-1,3 glycosidic bonds but not on substrates containing only β-1,4- or β-1,6-glycosidic bonds. Interestingly, compared to the activity of this enzyme towards carboxymethyl (CM)-pachyman containing only β-1,3-glycosidic bonds, its activity towards barley β-glucan containing both β-1,3-glycosidic and β-1,4-glycosidic bonds was increased by 64.72 %,, its activity towards laminarin containing both β-1,3-glycosidic and β-1,6-glycosidic bonds was decreased by 50.83 %. In addition, ENG16A has a higher Km value and Vmax for barley β-glucan than laminarin, which may be related to the fact that barley β-glucan contains mainly β-1,4-glycosidic bonds mixed with a few β-1,3-glycosidic bonds, whereas laminarin mainly contains β-1,3-glycosidic bonds with a few β-1,6-branched glucose residues. The adjacent β-1,4-glycosidic bond promotes ENG16A to hydrolyse β-1,3-glycosidic bonds, leading to an increased Vmax; the nearby β-1,6-glycosidic bonds inhibited its hydrolysis of β-1,3-glycosidic bonds, resulting in a decreased Vmax. This property is suggested to be related to the mechanism that C. cinerea uses to degrade and utilize hemicellulose in straw medium and to protect its cell wall during the mycelium growth stage.

•Native wall extension activity is located exclusively in apical region of stipes.•Wall extension profile is consistent with distribution of elongation growth in stipe.•A snail expansin-like protein induces stipe wall extension in a nonhydrolysis way.•Enzymic hydrolysis or turgor is not central to the mechanism of stipe elongation.Expansin proteins extend plant cell walls by a hydrolysis-free process that disrupts hydrogen bonding between cell wall polysaccharides. However, it is unknown if this mechanism is operative in mushrooms. Herein we report that the native wall extension activity was located exclusively in the 10 mm apical region of 30 mm Flammulina velutipes stipes. The elongation growth was restricted also to the 9 mm apical region of the stipes where the elongation growth of the 1st millimetre was 40-fold greater than that of the 5th millimetre. Therefore, the wall extension activity represents elongation growth of the stipe. The low concentration of expansin-like protein in F. velutipes stipes prevented its isolation. However, we purified an expansin-like protein from snail stomach juice which reconstituted heat-inactivated stipe wall extension without hydrolytic activity. So the previous hypotheses that stipe wall extension was resulted from hydrolysis of wall polymers by enzymes or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure are challenged. We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting noncovalent bonding between glucan chains or chitin chains.

Transforming naringin using the mycelium of Trichoderma harzianum CGMCC 1523 produces two metabolites, 3′,4′,5,7-tetrahydroxy flavanone-7-rhamnoglucoside (3′-OHN) and 3′,4′,5′,5,7-pentahydroxy flavanone-7-rhamnoglucoside (3′,5′-DOHN), both of which were characterized by ESI–MS, 1H NMR and 13C NMR analyses. The time course of the biotransformation by T. harzianum showed that 3′-OHN and 3′,5′-DOHN appeared simultaneously at 6 h, and the conversion yield (32.6%) of 3′,5′-DOHN was higher (10.6%) than that of 3′-OHN at 56 h. The optimal biotransformation temperature was 30 °C, the optimal pH was 5.0, and the optimal concentration of naringin was 400 mg/l. The bigger volume of biotransformation mixture and lower shaking speed did not favor hydroxylation reactions. The radical scavenging activity of naringin at 2000 μM was 11.1%, whereas activity of 3′-OHN at 100 μM could reach 38.4%, which is 68.6 times more than naringin. Antioxidative activity of 3′,5′-DOHN was increased 13.5% at 100 μM compared to 3′-OHN.

•A puerarin-glycosylating strain of Lysinibacillus fusiformis was isolated.•Two glycosides of puerarin were produced.•Puerarin-7-O-fructoside has improved water solubility and antioxidant activity.•The strain is organic solvent-tolerant.•Under optimal conditions, the conversion rate of puerarin reached 97.6 ± 2.3%.A bacterial strain able to glycosylate the plant natural product puerarin was isolated from local soil in Nanjing, China. It was identified as Lysinibacillus fusiformis, and deposited in China General Microbiological Culture Collection (CGMCC) under accession number 4913. Incubation of this strain with puerarin led to efficient production (91.6% conversation rate) of puerarin-7-O-fructoside, a derivative that possesses improved water solubility and antioxidant activity. A minor product puerarin-7-O-isomaltoside was also produced in small amounts, with a conversion rate of less than 1% after 48-h reaction. Both products were characterized based on the spectral data. Among the four tested sugars, sucrose (92.6% conversion rate of puerarin) is the best glycosyl donor for L. fusiformis CGMCC 4913, followed by maltose (39.8% conversion rate of puerarin), while glucose and fructose are not appropriate donors for this biotransformation process. L. fusiformis CGMCC 4913 can survive in the presence of 10% (v/v) organic solvents such as methanol, ethanol, toluene, cyclohexane, and dimethyl sulfoxide. The biotransformation efficiency of puerarin was increased 2-fold in the presence of 10% ethanol at 12 h compared to the transformation solution without ethanol. The optimum pH and substrate concentration are 8.0 and 4 g/L, respectively. Under the optimal conditions, the final conversion rate of puerarin reached 97.6 ± 2.3% at 48 h in the presence of 10% ethanol. Therefore, L. fusiformis CGMCC 4913 represents a new and efficient biocatalyst for the biotransformation of puerarin.