The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest K m and highest k cat/K m value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of k cat/K m) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.

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The compound 4-vinyl guaiacol (4-VG) is highly valued and widely applied in the pharmaceutical, cosmetic, and food industries. The bioproduction of 4-VG from ferulic acid (FA) by non-oxidative decarboxylation using phenolic acid decarboxylases is promising but has been hampered by low conversion yields and final product concentrations due to the toxicities of 4-VG and FA. In the current study, a new phenolic acid decarboxylase (BaPAD) was characterized from Bacillus atrophaeus . The BaPAD possessed excellent catalytic activity and stability in various organic solvents. Whole Escherichia coli cells harboring intracellular BaPAD exhibited greater tolerances to FA and 4-VG than those of free BaPAD. A highly efficient aqueous-organic biphasic system was established using 1-octanol as the optimal organic phase for whole-cell catalysis. In this system, a very high concentration (1580 mM, 237.3 g/L) of 4-VG was achieved in a 2 L working volume bioreactor, and the molar conversion yield and productivity reached 98.9% and 18.3 g/L/h in 13 h, respectively.

The spores of arbuscular mycorrhizal fungi (AMF) form a unique microhabitat that is suitable for the colonization by many species of bacteria. The aim of the current study was to analyze the bacterial communities associated with the surface of spores of the AMF species Gigaspora margarita MAFF 520054 and Gigaspora rosea JP1. The two AMF species were propagated with tobacco (Nicotiana tabacum) grown in a mixture of sand and soil. In another experiment, G. margarita was propagated with tobacco or alfalfa (Medicago sativa) grown in vermiculite or a mixture of sand and soil. The bacterial community composition of the new-formed spores and sand/soil substrate was analyzed using PCR of 16S rDNA fragments and denaturing gradient gel electrophoresis (DGGE). Clustering analysis revealed that the bacterial communities on the surface of G. margarita spores was different form that in the substrate or on the surface of the G. rosea spores, and both the host plant and the substrate could influence the composition of spore-associated bacterial populations of the G. margarita. Sequence analysis of the major DGGE bands of G. margarita spore samples revealed that most of the bacterial sequences were affiliated with the phyla Proteobacteria (Azospirillum,

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BACKGROUND4‐vinyl guaiacol (4‐VG) is a high value‐added product widely used in the cosmetic, pharmaceutical, and chemical industries. The practical bioproduction of 4‐VG using phenolic acid decarboxylases has been limited by its relatively high biocatalyst cost and low yield and product concentration.RESULTSIn the present study, high‐cell density cultivation was employed to improve the activity and production of phenolic acid decarboxylase from Bacillus licheniformis (BLPAD) in recombinant Escherichia coli. The factors influencing enzyme production in E. coli such as the induction point and temperature for induction and feeding strategies were optimized. The highest BLPAD activity (531 U mL‐1) and productivity (20.4 U mL‐1 h‐1), respectively, were achieved in a 5 L bioreactor using a glucose exponential feeding strategy with isopropyl β‐D‐thiogalactoside (IPTG) as inducer. Furthermore, a high BLPAD production level (512 U mL‐1) was achieved using lactose as an inducer and continuous lactose feeding. Using a biphasic emulsion system with equal volumes (1 L) of cyclohexane as a organic solvent and a substrate fed‐batch strategy, the concentration and conversion yield of 4‐VG reached 129.9 g L‐1 (85.6%) in a 5 L bioreactor by whole‐cell biocatalysis, which is the highest reported to date.CONCLUSIONThis study describes a strategy for large‐scale 4‐VG bioproduction using the biocatalytic method. © 2018 Society of Chemical Industry

XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency (k cat/K m) towards beechwood xylan than wild-type and 3.0-fold more thermostability (t 1/2 [55°C]). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life (t 1/2) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS.