Insight into broad substrate specificity and synergistic contribution of a fungal α-glucosidase in Chinese Nong-flavor daqu.

BACKGROUND: Chinese Nong-favor daqu, the presentative liquor starter of Baijiu, has been enriched with huge amounts of enzymes in degrading various biological macromolecules by openly man-made process for thousand years. According to previous metatranscriptomics analysis, plenty of α-glucosidases were identified to be active in NF daqu and played the key role in degrading starch under solid-state fermentation. However, none of α-glucosidases was characterized from NF daqu, and their actual functions in NF daqu were still unknown. RESULTS: An α-glucosidase (NFAg31A, GH31-1 subfamily), the second highest expressed α-glucosidases in starch degradation of NF daqu, was directly obtained by heterologous expression in Escherichia coli BL21 (DE3). NFAg31A exhibited the highest sequence identities of 65.8% with α-glucosidase II from Chaetomium thermophilum, indicating its origin of fungal species, and it showed some similar features with homologous α-glucosidase IIs, i.e., optimal activity at pH ~ 7.0 and litter higher temperature of 45 ℃, well stability at 41.3 ℃ and a broad pH range of pH 6.0 to pH 10.0, and preference on hydrolyzing Glc-α1,3-Glc. Besides this preference, NFAg31A showed comparable activities on Glc-α1,2-Glc and Glc-α1,4-Glc, and low activity on Glc-α1,6-Glc, indicating its broad specificities on α-glycosidic substrates. Additionally, its activity was not stimulated by any of those detected metal ions and chemicals, and could be largely inhibited by glucose under solid-state fermentation. Most importantly, it exhibited competent and synergistic effects with two characterized α-amylases of NF daqu on hydrolyzing starch, i.e., all of them could efficiently degrade starch and malto-saccharides, two α-amylases showed advantage in degrading starch and long-chain malto-saccharides, and NFAg31A played the competent role with α-amylases in degrading short-chain malto-saccharides and the irreplaceable contribution in hydrolyzing maltose into glucose, thus alleviating the product inhibitions of α-amylases. CONCLUSIONS: This study provides not only a suitable α-glucosidase in strengthening the quality of daqu, but also an efficient way to reveal roles of the complicated enzyme system in traditional solid-state fermentation. This study would further stimulate more enzyme mining from NF daqu, and promote their actual applications in solid-state fermentation of NF liquor brewing, as well as in other solid-state fermentation of starchy industry in the future.

Fungal Biomarkers in Traditional Starter Determine the Chemical Characteristics of Turbid Rice Wine from the Rim of the Sichuan Basin, China.

The fungal community in Qu plays a key role in the formation of turbid rice wine (TRW) style. The Sichuan Basin and its surrounding areas have become one of the main TRW production regions in China; however, the fungal community in Qu and how they affect the characteristics of TRW remain unknown. Therefore, this study provided insight into the fungal biomarkers in Qu from Guang'an (GQ), Dazhou (DQ), Aba (AQ), and Liangshan (LQ), as well as their relationships with compounds in TRW. The main biomarkers in GQ were Rhizopus arrhizus, Candida glabrata, Rhizomucor pusillus, Thermomyces lanuginosus and Wallemia sebi. However, they changed to Saccharomycopsis fibuligera and Mucor indicus in DQ, Lichtheimia ramose in AQ, and Rhizopus microsporus and Saccharomyces cerevisiae in LQ. As a response to fungal biomarkers, the reducing sugar, ethanol, organic acids, and volatile compounds were also changed markedly in TRWs. Among important volatile compounds (VIP > 1.00), phenethyl alcohol (14.1-29.4%) was dominant in TRWs. Meanwhile, 3-methyl-1-butanol (20.6-56.5%) was dominant in all TRWs except that fermented by GQ (GW). Acetic acid (29.4%) and ethyl palmitate (10.1%) were dominant in GW and LW, respectively. Moreover, GQ biomarkers were positively correlated with acetic acid and all unique important volatile compounds in GW. DQ biomarkers had positive correlations with unique compounds of acetoin and ethyl 5-chloro-1,3,4-thiadiazole-2-carboxylate in DW. Meanwhile, the AQ biomarkers were positively correlated with all AW unique, important, and volatile compounds. Although there were not any unique volatile compounds in LW, 16 important volatile compounds in LW were positively related to LQ biomarkers. Obviously, biomarkers in different geographic Qu played vital roles in the formation of important volatile compounds, which could contribute specific flavor to TRWs. This study provided a scientific understanding for future efforts to promote the excellent characteristics of TRW by regulating beneficial fungal communities.

Nitric oxide enhances resistance of Pleurotus eryngii to cadmium stress by alleviating oxidative damage and regulating of short-chain dehydrogenase/reductase family.

The function and mechanism of nitric oxide (NO) in regulating Pleurotus eryngii biological response to cadmium (Cd) stress was evaluated by using anti-oxidation and short-chain dehydrogenase/reductase (SDR) family analysis. The fresh biomass of P. eryngii mycelia sharply decreased after treatment with 50 µM Cd; the lipid peroxidation and H2O2 accumulation in P. eryngii were found responsible for it. Proper exogenous supply of NO (150 µM SNP) alleviated the oxidative damage induced by Cd stress in P. eryngii, which reduced the accumulation of thiobarbituric acid reactive substances (TBARS) and H2O2. The activities of antioxidant enzymes (superoxide dismutase, peroxidase) were significantly increased to deal with Cd stress when treated with SNP (150 µM), and the content of proline was also closely related to NO-mediated reduction of Cd toxicity. Moreover, SDR family members were widely involved in the response to Cd stress, especially PleSCH70 gene was observed for the first time in participating in NO-mediated enhancement of Cd tolerance in P. eryngii. Taken together, this study provides new insights in understanding the tolerance mechanisms of P. eryngii to heavy metal and lays a foundation for molecular breeding of P. eryngii to improve its tolerance to environmental stress.

Biochemical and synergistic properties of a novel alpha-amylase from Chinese nong-flavor Daqu.

BACKGROUND: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. RESULTS: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. CONCLUSIONS: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

Uncovering key residues responsible for the thermostability of a thermophilic 1,3(4)-ß-d-glucanase from Nong flavor Daqu by rational design.

Fungal 1,3(4)-ß-D-glucanases were usually applied in brewing and feedstuff industries, however, the thermostability limits the most their application. The characterized 1,3(4)-ß-D-glucanase (NFEg16A) from Chinese Nong-flavor (NF) Daqu showed the highest thermostability among GH16 fungal 1,3(4)-ß-D-glucanases, with half-lives of thermal inactivation (t1/2) of 44.9 min at 90 °C, so multiple rational designs were used to identify the key residues for its thermostability. Based on protein sequence and 3D structure analyses around the catalytic regions. Nine site-mutants were constructed, among which N173Y and S187A were identified as the most thermotolerant and thermolabile ones, with t1/2 values of 61 min and 14.0 min at 90 °C, respectively. Therefore, N173 and S187 were then selected as "hotspots" for site-saturation mutagenesis. Interestingly, most of the N173 and S187 variants exhibited a similar thermostability to that of N173Y and S187A, respectively, confirming their different roles in the thermostability of NFEg16A. In addition, each S187A and its surrounding substitutions (D144 N and T164 N) was independently detrimental to the thermostability of NFEg16A, since the t1/2 (90 °C) of S187A, D144 N and T164 N were 14.0 min, 20.6 min and 27.2 min, respectively. Surprisingly, combinatorial substitution of S187A with D144 N or T164 N showed positive effects on the thermostability, with the increase of t1/2 (90 °C) to 30.9 min and 63.5 min for S187A-D144 N and S187A-T164 N, respectively. More importantly, S187A-T164 N showed higher thermostability than that of wild type. In short, we successfully identified two key sites and their surrounding residues in response to the thermostability of NFEg16A and further improved its thermostability by several rational designs. These findings could be used for the protein engineering of homologous 1,3(4)-ß-D-glucanases, as well as other enzyme family members with high similarities.

Unraveling the Contribution of High Temperature Stage to Jiang-Flavor Daqu, a Liquor Starter for Production of Chinese Jiang-Flavor Baijiu, With Special Reference to Metatranscriptomics.

Jiang-flavor (JF) daqu is a liquor starter used for production of JF baijiu, a well-known distilled liquor in China. Although a high temperature stage (70°C) is necessary for qualifying JF daqu, little is known regarding its active microbial community and functional enzymes, along with its role in generating flavor precursors for JF baijiu aroma. In this investigation, based on metatranscriptomics, fungi, such as Aspergillus and Penicillium, were identified as the most active microbial members and 230 carbohydrate-active enzymes were identified as potential saccharifying enzymes at 70°C of JF daqu. Notably, most of enzymes in identified carbohydrate and energy pathways showed lower expression levels at 70°C of JF daqu than those at the high temperature stage (62°C) of Nong-flavor (NF) daqu, indicating lowering capacities of saccharification and fermentation by high temperature stage. Moreover, many enzymes, especially those related to the degradation of aromatic compounds, were only detected with low expression levels at 70°C of JF daqu albeit not at 62°C of NF daqu, indicating enhancing capacities of generating special trace aroma compounds in JF daqu by high temperature stage. Additionally, most of enzymes related to those capacities were highly expressed at 70°C by fungal genus of Aspergillus, Coccidioides, Paracoccidioides, Penicillium, and Rasamsonia. Therefore, this study not only sheds light on the crucial functions of high temperature stage but also paves the way to improve the quality of JF baijiu and provide active community and functional enzymes for other fermentation industries.

Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method.

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-ß-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-ß-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60 °C and highly stable (>75% residual activity) at pH 3-8 and temperature 30-90 °C. The activity of NFEg16A was strongly inhibited by 10 mM Fe3+ and Hg2+. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70 °C and had higher half-lives of 3.4 h and 1.4 h at 80 °C and 90 °C, respectively. Its specific activity was 85.3 U/mg on barley ß-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.