Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0–7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2′-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.

Laccases have potential applications in detoxification of lignocellulosic biomass after thermochemical pretreatment and production of value-added products or biofuels from renewable biomass. However, their application in large-scale industrial and environmental processes has been severely thwarted by the high cost of commercial laccases. Therefore, it is necessary to identify new laccases with lower cost but higher activity to detoxify lignocellulosic hydrolysates and better efficiency to produce biofuels such as bioethanol. Laccases from Ganoderma lucidum represent proper candidates in processing of lignocellulosic biomass. G. lucidum 77002 produces three laccase isoenzymes with a total laccase activity of 141.1 U/mL within 6 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A new isoenzyme named Glac15 was identified, purified, and characterized. Glac15 possesses an optimum pH of 4.5 to 5.0 and a temperature range of 45°C to 55°C for the substrates tested. It was stable at pH values ranging from 5.0 to 7.0 and temperatures lower than 55°C, with more than 80% activity retained after incubation for 2 h. When used in bioethanol production process, 0.05 U/mL Glac15 removed 84% of the phenolic compounds in prehydrolysate, and the yeast biomass reached 11.81 (optimal density at 600 nm (OD600)), compared to no growth in the untreated one. Addition of Glac15 before cellulase hydrolysis had no significant effect on glucose recovery. However, ethanol yield were improved in samples treated with laccases compared to that in control samples. The final ethanol concentration of 9.74, 10.05, 10.11, and 10.81 g/L were obtained from samples containing only solid content, solid content treated with Glac15, solid content containing 50% prehydrolysate, and solid content containing 50% prehydrolysate treated with Glac15, respectively. The G. lucidum laccase Glac15 has potentials in bioethanol production industry.

Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety.

The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored.

Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0–7.5 and 25–50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.