Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design.

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate (CuSO4). The best conditions obtained from screening experiments in shaken flasks using tomato juice, CuSO4, and soybean oil were integrated in an experimental design. Enhanced levels of tomato juice as the medium, CuSO4 and soybean oil as inducers (36.8% (v/v), 3 mmol/L, and 1% (v/v), respectively) were determined for 10 L stirred tank bioreactor runs. This combination resulted in laccase titer of 143,000 IU/L (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), pH 3.0), which represents the highest activity so far reported for P. sanguineus in a 10-L fermentor. Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20,000 IU/L, whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times. Based on a partial characterization, the laccases of this strain are especially promising in terms of thermostability (half-life of 6.1 h at 60 °C) and activity titers.

Screening for thermotolerant ligninolytic fungi with laccase, lipase, and protease activity isolated in Mexico.

The State of Hidalgo (Mexico) has a large area of forests known as the Huasteca Hidalguense, with a large variety of microorganisms inhabiting it. They represent an important resource from the ecological and technological point of view because they can be used in a broad variety of industrial processes. Due to the climatic conditions of this region, fungi inhabiting it must be thermophile or, at least, thermotolerant, as temperatures can be higher than 45°C in the summer, declining to 20°C in the winter. Use of ligninolytic fungi relies on their capacity to produce enzymes of industrial interest, a topic that has been under continuous research by academic and industrial investigators. Among the most important enzymes are proteases that are widely used due to their biotechnological applications with a high economic impact. Other enzymes, laccases, peroxidases, and lipases are of interest for the industries of the state of Hidalgo, especially in the textile industry, specifically in effluent processing. Fungi (n=156) were collected in the Huasteca Hidalguense, of which 100 were isolated in potato-dextrose-agar covered plates and maintained in tilted tubes. Afterwards, enzymatic activity (laccase, protease and lipase) was determined in the plates. The purpose was to select those fungi with the highest potential for biotechnological applications. Fungi generally grew at either 30°C or 37°C, and for some isolates enzymatic activities were detected at this higher temperature. Results are presented as the relation between enzymatic activity and growth rate: 60 fungi presented laccase activity, 49 had lipase activity, and none had protease activity. In most cases, enzymatic activity was higher than the growth rate, indicating that the isolated fungi have a great biotechnological potential. Statistical analysis revealed that isolates 31 (Trametes) and 8.1 (unidentified) have a larger potential to be studied as laccase-producing fungi. On the other hand, isolates 144.2 (Fomes), 154 (Trametes), and 147.2 (Pycnoporus) are of interest as lipase activity producers, an activity scarcely studied in this type of microorganisms.

Production and characterization of a novel laccase with cold adaptation and high thermal stability from an isolated fungus.

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml(-1), which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0 degrees C but also a high thermostable enzyme. The half-lives at 60, 70 and 80 degrees C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.