Utilization of used fungus-growing materials of Gastrodia elata / 中国中药杂志

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Influence of Temperature on the Bacterial Community in Substrate and Extracellular Enzyme Activity of Auricularia cornea

Temperature is an important environmental factor that can greatly influence the cultivation of Auricularia cornea. In this study, lignin peroxidase, laccase, manganese peroxidase, and cellulose in A. cornea fruiting bodies were tested under five different temperatures (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C) in three different culture periods (10 days, 20 days and 30 days). In addition, the V4 region of bacterial 16S rRNA genes in the substrate of A. cornea cultivated for 30 days at different temperatures were sequenced using next-generation sequencing technology to explore the structure and diversity of bacterial communities in the substrate. Temperature and culture days had a significant effect on the activities of the four enzymes, and changes in activity were not synchronized with changes in temperature and culture days. Overall, we obtained 487,694 sequences from 15 samples and assigned them to 16 bacterial phyla. Bacterial community composition and structure in the substrate changed when the temperature was above 35 °C. The relative abundances of some bacteria were significantly affected by temperature. A total of 35 genera at five temperatures in the substrate were correlated, and 41 functional pathways were predicted in the study. Bacterial genes associated with the membrane transport pathway had the highest average abundance (16.16%), and this increased at 35 °C and 40 °C. Generally, different temperatures had impacts on the physiological activity of A. cornea and the bacterial community in the substrate; therefore, the data presented herein should facilitate cultivation of A. cornea.

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40­50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h. Conclusions: The recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.

Cloning, expression and characterization of the endoglucanase gene from Bacillus subtilis UMC7 isolated from the gut of the indigenous termite Macrotermes malaccensis in Escherichia coli

Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose.

Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus)

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (beta-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.

Variation of substance content during growth cycle of cultured Ganoderma lucidum and its mechanism / 中草药

Objective: To study the variation of the substance content in the different fractions of matrix during the growth cycle of Ganoderma lucidum, and to explore the mechanisms by investigating the change regulation of extracellular enzyme activity in the matrix of G. lucidum. Methods: G. lucidum was adopted by bag cultivation with sterilized raw materials, the growth period was divided into eight stages, and the matrix was splided in four equal portions. The variation of the contents of total sugar, reducing sugar, water-soluble protein, and free amino acids in different fractions of the matrix was studied, and the variation of activities of lignin enzymes (laccase and manganese peroxidase), cellulase (carboxymethyl cellulase, hemicellulase, and filter paper enzyme), and acidic protease at different fractions of the matrix were studied in order to elucidate the mechanisms. Results: During the growth period of G. lucidum, the content of total sugar at different fractions of the matrix decreased, and the content of reducing sugar increased before the growth of primordium, then decreased after the growth of primordium. The contents of water-soluble proteins showed increasing tendency at different fractions of the matrix, and the content of amino acid decreased before sackful of mycelium, but increased during the stage of bud and reached the peak, then decreased again. The variation of laccase activity showed the shape of U, and the lowest activity was appeared in the primordium period. The activity of manganese peroxidase was slowly increased during vegetative growth period, and decreased after sackful of mycelium. The activities of cellulase enzyme and acid protease were totally increased. Conclusion: In the growth cycle of G. lucidum, the substance contents in different fractions of matrix present different change tendency with the growth and development of G. lucidum; The extracellular enzyme activity of G. lucidum could affect the content of substance in the matrix, and there is a certain correlation between the activity of extracellular enzyme and material content in matrix of G. lucidum.

A New Record of Penicillium antarcticum from Marine Environments in Korea

During a survey of marine fungi from the waters surrounding Jeju Island, Korea, several Penicillium strains were isolated from seawater and marine sponges. Based on morphological characteristics and phylogenetic analyses of the internal transcribed spacer and RNA polymerase subunit II, four strains were identified as Penicillium antarcticum, a fungus that, to the best of our knowledge, had not been previously reported in Korea. Here, we provide detailed descriptions of the morphological characteristics and extracellular enzyme activities of the four strains.

Enzyme Activity of Cenococcum geophilum Isolates on Enzyme-specific Solid Media

Enzyme activities of Cenococcum geophilum isolates were examined on enzyme-specific solid media. Deoxyribonuclease, phosphatase, and urease were detected in all isolates, whereas cellulase was not detected in any of the isolates. Variations in enzyme activities of amylase, caseinolysis, gelatinase, lipase, and ribonuclease were observed among isolates.

Extracellular Enzyme Activities of the Monokaryotic Strains Generated from Basidiospores of Shiitake Mushroom

To obtain basic information on the biochemical property of basidiospores of shiitake mushroom (Lentinula edodes), the ability of producing extracellular enzyme was assessed using a chromogenic plate-based assay. For the aim, amylase, avicelase, beta-glucosidase, CM-cellulase, pectinase, proteinase, and xylanase were tested against monokaryotic strains generated from forty basidiospores of two different parental dikaryotic strains of shiitake mushroom, Sanjo-101Ho and Sanjo-108Ho. These two parental strains showed different degree of extracellular enzyme activity. No identical patterns of the degree of enzyme activity were observed between monokaryotic strains and parental strains of the two shiitake cultivars. The degree of extracellular enzyme activity also varied among monokaryotic strains of the two shiitake cultivars. Our results showed that dikaryotic parental strains of shiitake mushroom produce monokaryotic basidiospores having very diverse biochemical properties.

Preliminary Characterization of Keratinolytic Enzyme of Aspergillus flavus K-03 and Its Potential in Biodegradation of Keratin Wastes

Aspergillus flavus K-03 isolated from poultry forming soil in Korea was studied for its ability to produce extracellular proteases on basal medium containing 2% (w/v) chicken feathers. The fungus was observed to be a potent producer of such enzymes. Keratinolytic enzyme secretion was the best at 15 days of incubation period at pH 9 and temperature 40degrees C. No relationship existed between the enzyme yield and increase of biomass. Enzyme production was suppressed by exogenous sugars in descending order arabinose>maltose>mannose>fructose. But glucose did not influence the enzyme activity. The keratinolytic enzyme released by the fungus demonstrated the ability to decompose keratin substrates as chicken feather when exogenous glucose was present. The keratinolytic activity was inhibited by HgCl2 and serine-protease inhibitors such as phenymethylsulfonyl fluoride (100%), chymostain (88%), crystalline soybean trypsin inhibtor (80%), antipain (45%) and aprotinin (40%), and was not by cystein-protease and aspartyl-protease inhibitors. The enzyme activity is only partially inhibited by metallo-protease inhibitor. Thus, the enzyme secreted by A. flavus K-03 belongs to the alkaline serine-type protease.

STUDY ON THE PROPERTIES OF POLY(?-HYDROXYBUTYRATE)DEPOLYMERASE / 微生物学通报

Three strains having degrading poly(?-hydroxybutyrate)(PHB) activity were isolated from activated sludge of different ecological environments and areas,named DS9701, DS9710 and DS9713.The properties of PHB depolymerase produced by DS9701, DS9710 and DS9713 were studied. All the PHB depolymerases are extracellular enzyme and are induced enzyme. The time that enzyme activities of the PHB depolymerases reach the maximum is 96 hours after inoculation. The apparent optimal temperature range for crude enzymes extract is 40℃～45℃.

DEGRADATION AND TRANSFORMATION OF COTTON SEED HULLS BY COPRINUS COMATUS / 微生物学通报

Coprinus comatus cultivated on cotton seed hull medium decomposed lignocellulose straggly and was high of absolute biological efficiency. Lignocellulose is the main carbon source for the fruiting stage of the fungus. There existed the positive correlation between the degradation rates of the cellulose and hemicellulose in the medium and the activities of extracellular CMCase (carboxymenthelcel lulase), FPase (filter paper cellulase) and HCase (hemicellu-lase), there also existed the positive correlation between the degradation rate of the lignin in the medium and the activity of extracellular laccase, but no correlation between the degradatio rate of the lignin in the medium and the activity of peroxidase. The activity of extracellular amylase was comparatively high at mycelial growth stage, and the protease activity peek was at teh time when the fruitbody matured.