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Black rice contains a variety of bioactive substances that contribute to the high nutritional value of black rice wine (BRW). However, the dense bran layer of black rice retards the fermentation rate and reduces the dissolution of active components. Hence, this study aims to investigate the effects of pre-gelatinization (PG) before cooking and cellulase (CE) addition during fermentation on the fermentation performance of BRW and its antioxidant activity.PG combined with CE treatments (PGCE) increases the alcohol content, free amino acid content, volatile flavor content and total antioxidant activity of BRW by 90.81%, 15.36%, 38.05% and 19.56%, respectively, compared with the control group. Scanning electron microscopy, low-field nuclear magnetic resonance and texture properties analysis indicate that PG treatment increases gelatinization degree of starch during cooking, decreases bound water content in cooked black rice and promotes unbound water release. CE destroys the aleurone layer structure, facilitates the release of unbound water and the exposure of rice starch, thus increasing the reaction area and extravasation content significantly, which is beneficial to microbial growth and fermentation. Incomplete aleurone layer also promotes the dissolution of anthocyanins, phenols and other active substances, increasing the antioxidant activities of BRW.PG and CE treatments reduce the fermentation time and improve the quality of BRW by destroying the black rice structure. © 2022 Society of Chemical Industry.

EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (K

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

The utilization of sugarcane bagasse (SB) in fermentation requires pretreatment processes to render fermentable components available to microorganisms. Pretreatment by using ionic liquids (ILs) is considered promising but the high cost is an impediment in its adoption, therefore, a mixture of IL pretreated and untreated SB was utilized to obtain bacterial multienzyme under solid-state fermentation (SSF). Bacillus aestuarii UE25, a thermophilic strain was utilized for that purpose. Fermentation conditions were optimized by adopting a central composite design. The model showed a good correlation between the predicted and the experimental values for amylase, xylanase, endoglucanase, and β-glucosidase. Volumetric and specific productivity of xylanase (4580 IU ml-1 h-1 , 244.25 IU mg-1 substrate, and 50 IU mg-1 protein) were higher than the other enzymes. Changes in lignin content and reduced cellulose crystallinity due to IL pretreatment, followed by fermentation, were visualized by scanning electron microscopy, Fourier transform infrared spectroscopy, and Nuclear magnetic resonance. The strategy adopted by utilizing a mixture of IL pretreated and untreated SB under SSF proved promising to obtain high titers of different enzymes simultaneously. Since the bacterial strain used is thermophilic, therefore, the multienzyme can find its application in commercial processes which are carried out at high temperatures.

Summary Exorbitant outputs of waste xylose mother liquor (WXML) and corncob residue from commercial‐scale production of xylitol create environmental problems. To reduce the wastes, a Saccharomyces cerevisiae strain tolerant to WXML was conferred with abilities to express the genes of xylose reductase, a xylose‐specific transporter and enzymes of the pentose phosphate pathway. This strain showed a high capacity to produce xylitol from xylose in WXML with glucose as a co‐substrate. Additionally, a simultaneous saccharification and fermentation (SSF) process was designed to use corncob residues and cellulase instead of directly adding glucose as a co‐substrate. Xylitol titer and the productivity were, respectively, 91.0 g l‐1 and 1.26 ± 0.01 g l‐1 h‐1 using 20% WXML, 55 g DCW l‐1 delignified corncob residues and 11.8 FPU gcellulose‐1 cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process.

β-glucosidases (BGLs) belong to the group of enzymes of cellulases and acts in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of β-glucosidase from Moniliophthora perniciosa in the hydrolysis of pre-treated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein. This article is protected by copyright. All rights reserved.

P. eryngii and P. ostreatus protein expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability. Co‐regulated carbohydrate‐active enzymes were associated with selective ligninolysis of ramie stalks., Summary In the present research, Phanerochaete chrysosporium and Irpex Lacteus simultaneously degraded lignin and cellulose in ramie stalks, whereas Pleurotus ostreatus and Pleurotus eryngii could depolymerize lignin but little cellulose. Comparative proteomic analysis of these four white‐rot fungi was used to investigate the molecular mechanism of this selective ligninolysis. 292 proteins, including CAZymes, sugar transporters, cytochrome P450, proteases, phosphatases and proteins with other function, were successfully identified. A total of 58 CAZyme proteins were differentially expressed, and at the same time, oxidoreductases participated in lignin degradation were expressed at higher levels in P. eryngii and P. ostreatus. Enzyme activity results indicated that cellulase activities were higher in P. chrysosporium and I. lacteus, while the activities of lignin‐degrading enzymes were higher in P. eryngii and P. ostreatus. In addition to the lignocellulosic degrading enzymes, several proteins including sugar transporters, cytochrome P450 monooxygenases, peptidases, proteinases, phosphatases and kinases were also found to be differentially expressed among these four species of white‐rot fungi. In summary, the protein expression patterns of P. eryngii and P. ostreatus exhibit co‐upregulated oxidoreductase potential and co‐downregulated cellulolytic capability relative to those of P. chrysosporium and I. lacteus, providing a mechanism consistent with selective ligninolysis by P. eryngii and P. ostreatus.

In a previous study, we reported that ethylene treatment facilitated boil‐peeling in persimmons and in several other fruits; however, the mechanism underlying the facilitating effect of ethylene was not examined in detail. Thus, in this study, we investigated the effect of ethylene treatment on the peel characteristics of persimmons, that facilitated boil‐peeling, using chemical, genomic, and histochemistry analyses. The results of the study showed that the ethylene‐related genes, DK‐ACS1 and DK‐ACO2, and the pectinase‐active gene DKPG were not expressed, even though a minor increase in ethylene generation was observed after ethylene treatment. Conversely, significant accumulation of toluidine blue O and ruthenium red dyes were observed in the sarcocarp and exocarp of the fruits, indicating an increase in the quantity of polysaccharides, including pectic substances, at the site. The results also indicate that the increased cellulase activity observed in the pericarp of the fruits may be due to the aging of the fruits, and not necessarily as a result of ethylene treatment. Furthermore, ethylene treatment increased the quantity of polysaccharides, including pectic substances, directly below the pericarp, which caused the dissolution of the site, resulting in peeling. This study provides new insights on the effect of ethylene on boil‐peeling in persimmons and provides a foundation for future research studying the effect of heat treatment in the peeling of fruits or tomato., Boil‐peeling in persimmons was possible because ethylene treatment increased the quantity of pectic substances in the pericarp of the fruit which resulted in the softening of the peel.

Background Wheat Qu has long been used as a fermentation starter to produce Huangjiu. Wheat Qu quality depends on its microbial community structure and the hydrolytic enzymes generated by the micro-organisms. Results Strain YF1 and YF2 were successfully screened as they exhibited high acidic protease (231.9 ± 1.4 U g-1 ) and cellulase (7.1 ± 0.6 U g-1 ) activities. Based on a morphological and sequence analysis of the internal transcribed spacer (ITS) gene, YF1 and YF2 were identified as Rhizopus oryzae and Aspergillus niger, respectively. Cooked wheat Qu was produced using mixed fungal starter fermentations with Aspergillus oryzae SU-16, YF1, and YF2. For Qu-making, the optimised conditions for fermentation time, water content, and inoculum size were 47.8 h, 69.4%, and 6.1%, respectively. Under these conditions, compared with single-strain cooked wheat Qu, enzyme activities of amylase, acidic protease, and cellulase increased by 27.4%, 657.1%, and 1,276.2%, respectively. Short peptides and free amino acids contents increased by 19.6% and 131.8%, respectively. This wheat Qu was used for Huangjiu brewing, and the alcohol content increased by approximately 14.6% because of the increased starch hydrolysis efficiency mainly attributed to its high enzyme activity. Conclusion Using mixed fungal strains as starter cultures may be an efficient strategy to improve wheat Qu quality, with great potential for application in industrial Huangjiu production. This article is protected by copyright. All rights reserved.

Consolidated bioprocessing (CBP) of cellulose is a cost-effective route to produce valuable biochemicals by integrating saccharification, fermentation and cellulase synthesis in a single step. However, the lack of understanding of governing factors of interdependent saccharification and fermentation in CBP eludes reliable process optimization. Here, we propose a new framework that synergistically couples population balances (to simulate cellulose depolymerization) and cybernetic models (to model enzymatic regulation of fermentation) to enable improved understanding of CBP. The resulting framework, named the unified cybernetic-population balance model (UC-PBM), enables simulation of CBP driven by coordinated control of enzyme synthesis through closed-loop interactions. UC-PBM considers two key aspects in controlling CBP: (1) heterogeneity in cellulose properties and (2) cellular regulation of competing cell growth and cellulase secretion. In a case study on Clostridium thermocellum, UC-PBM not only provides a decent fit with various exometabolomic data, but also reveals that: (i) growth-decoupled cellulase-secreting pathways are only activated during famine conditions to promote the production of growth substrates, and (ii) starting cellulose concentration has a strong influence on the overall flux distribution. Equipped with mechanisms of cellulose degradation and fermentative regulations, UC-PBM is practical to explore phenotypic functions for primary evaluation of microorganisms' potential for metabolic engineering and optimal design of bioprocess.

BACKGROUND: High‐efficiency saccharification technology is one of the bottlenecks of cellulosic bio‐hydrogen production. Cellulosic feedstocks saccharification currently performed by commercial cellulase, which is composed of different fungal cellulase. Compared with fungi, thermocellulosic bacteria represented by Ruminiclostridium thermocellum have a complete cellulase system, and a higher cellulase catalytic efficiency than fungi; however, R. thermocellum is susceptible to feedback inhibition by cellobiose, which limits the application of R. thermocellum on cellulosic bio‐hydrogen production. In this study, a strain named R. thermocellum M3, which is not subject to feedback inhibition by cellobiose, was used in the bio‐hydrogen production of cellulosic agricultural waste feedstocks to explore the feasibility of bacterial saccharification of cellulosic substrates for biological hydrogen production. RESULTS: Results of batch tests indicate that the combination of domestic sewage sludge and strain M3 promoted the hydrogen production for different lignin content feedstocks (rice straw: from 0.66 to 6.42 mmol H₂/g substrate; corn cob: from 0.61 to 5.55 mmol H₂/g substrate; pine wood waste: from 0.58 to 5.32 mmol H₂/g substrate), which were competitive with the combination of domestic sewage sludge and Trichoderma viride cellulase. Specific activity analysis indicates that compared with the addition of T. viride cellulase, the addition of strain M3 completed the cellulase system in sludge. CONCLUSION: Thermo‐anaerobic bacteria R. thermocellum M3 enhanced the hydrogen production of the consolidated bioprocessing (CBP) of raw lignocellulosic agricultural wastes and, more importantly, provided a promising solution for the CBP strategy in the industrial application of lignocellulose bioconversion. © 2021 Society of Chemical Industry

BACKGROUND The study aimed to evaluate the effect of Lactobacillus casei TH14, cellulase, and molasses combination fermented sugarcane bagasse (SB) as an exclusive roughage source in the total mixed ration (TMR) for mid-lactation 75% crossbred Holstein cows on feed intake, digestibility, ruminal ecology, milk yield and milk composition. Four multiparous mid-lactation crossbred (75% Holstein Friesian and 25% Thai native breed) dairy cows of 439 ± 16 kg body weight, 215 ± 5 days in milk and average milk yield 10 ± 2 kg d-1 were assigned to a 4 × 4 Latin square design. The unfermented SB (SB-TMR), SB fermented with cellulase and molasses (CM-TMR), SB fermented with L. casei TH14 and molasses (LM-TMR), and SB fermented with L. casei TH14, cellulase and molasses (LCM-TMR) were used as dietary treatments. RESULTS CM-TMR, LM-TMR and LCM-TMR significantly (P

Summary High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.

Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167 bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77 g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.

Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-β-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Resveratrol, a stilbene phytoalexin in plants, is believed to benefit human health. In this study, an optimized enzyme-assisted method was developed to extract the total content of trans-resveratrol (free or combined with glucose) in peanut seeds, followed by detection using high-performance liquid chromatography. The extraction process was optimized by Box-Behnken design and response surface methodology. The optimized enzyme concentration, digestion time, pH, and temperature were 3.02 g/L, 57.06 min, 5.88, and 51.05°C, respectively. Validation tests indicated that the experimental yield of trans-resveratrol was 0.183 ± 0.007 µg/g with a relative standard deviation of 3.87% (n = 5) under the optimal condition, which was closely agreed with the predicted value (0.182 µg/g). The recoveries obtained from the spiked samples were varied from 89.4 to 103.9%. Therefore, this study will provide a useful method for quantification of total trans-resveratrol in peanut seeds.

Introduction Rosmarinic acid is a bioactive compound with various pharmaceutical effects and applications. Objective This work developed a new approach for aqueous enzymatic extraction of rosmarinic acid from the leaves of Salvia officinalis. Methods Different enzymes (proteases and cellulase) were evaluated for their extraction activity. Response surface methodology (RSM) was subsequently employed to optimise the extraction conditions. Thin layer chromatography was also used to identify rosmarinic acid in the extract of S. officinalis. Results Among the tested enzymes, a Cellulase A and Protamex mixture (1:1, w/w) exhibited maximum effectiveness in the extraction. Through the use of RSM, the maximum rosmarinic acid content of 28.23 ± 0.41 mg/g was obtained with enzyme loading of 4.49%, water-to-sample ratio of 25.76 mL/g, temperature of 54.3°C, and extraction time of 2 h. Conclusion This study suggests that S. officinalis is a promising source of rosmarinic acid and aqueous enzymatic extraction is an efficient and ecofriendly method for extracting rosmarinic acid, with a short extraction time and without the contamination of a toxic solvent.

The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector-based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae PHAC1 or PKAR2 promoters. The eGFP reporter under control of PKAR2 was identified as the preferred combination due to its superior dynamic range and its greater sensitivity when measuring UPR induction in cellulase producing strains. To our knowledge, we show for the first time that significant UPR activation differences could consistently be observed for different cellulase candidate genes unlike previous RNA-based tests, which were unable to detect these differences. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them favorable for use in CBP yeasts.

The utilization of lignocellulosic biomass for biofuel production is a very important step in the transition towards to the circular economy. Within this process, the enzymatic hydrolysis are employed in a conversion of renewable biomass to a value-added product. The predominant polymer in lignocellulose is cellulose and enzymes involved in its degradation are cellulases. Those group of enzymes play a crucial role in biochemical degradation of recalcitrant nature of plant lignocellulose, so marketable demand for them is rapidly growing. A leading enzyme manufacturers, like Novozymes, AB Enzymes, DSM, Genencor DuPont, have already developed a highly efficient cellulose-digesting enzymes cellulases for industrial use. Over time, those enzyme preparations have been improved and novel enzymes innovations in the form of enzyme cocktails have been launched on the market. Consecutively, this great expansion maintains a constant need among researchers for isolating and selecting the novel microorganisms, more potent producers of industrially powerful enzymes.

Biotechnology & bioengineering 10, (2022). doi:10.1002/bit.28110, Published by Wiley, New York, NY [u.a.]

The solid-solid (immobilized cellulase-insoluble cellulose) phase cellulose hydrolysis reaction is significant in cellulosic biomass conversion processes but hindered because of its low efficiency. Herein, a smart temperature-pH dual-responsive material (D-N-N material) was prepared to be used as a carrier for cellulase recovery. This D-N-N material could undergo reversible and switchable transitions between solution, hydrogel, and solid phases. The following results were demonstrated: 1) the hydrolytic degree of this strategy could be as high as that of free cellulase in buffer solution; 2) the cellulase could be encapsulated into the D-N-N hydrogel without significant leaching and most of the cellulase activity was retained after recycling for at least 10 batches; and 3) more than 95 % of the glucose inside the hydrogel could be extracted during the hydrogel-solid transition within 1 h, which can assist in the high-efficiency separation of cellulase from glucose. The results suggested that this strategy provides a feasible platform for efficient cellulose hydrolysis and could be applied to other bio-derived reactions.