Efficient production of sophorose from glucose and its potentially industrial application in cellulase production.

Sophorose is the most effective inducer for cellulase production by Trichoderma reesei. Currently, the biosynthesis of sophorose is very inefficient, resulting in that unavailable for cellulase production in industry. In this study, CoGH1A, a multifunctional thermophilic glycoside hydrolase, was employed for sophorose production. Under the optimized conditions, the sophorose yield was 37.86 g/L with a productivity of 9.47 g/L/h which is by far the highest productivity. Meanwhile, the Fe3O4-CS-THP-CoGH1A nanoparticles were constructed to realize the recycling of CoGH1A. After 5 cycles of catalysis, Fe3O4-CS-THP-CoGH1A retained about 83.90 % enzyme activity. Finally, the mixtures of glucose and disaccharides (MGDC) obtained after being catalyzed by CoGH1A was used for cellulase production. As a result, the cellulase productivity achieved 188.38 FPU/L/h in 120 h. These results indicated that sophorose could be efficiently produced from glucose via transglycosylation by CoGH1A, making it possible to be industrially used as the inducer to improving the cellulase productivity.

Effect of incubation conditions of cellulase hydrolysis on mechanical pulp fibre morphology.

The mechanical pulp industry is diversifying through the manufacture of high-value paper products, such as microfibrillated cellulose. However, the development of fibre quality is still energy-intensive. Enzymatic hydrolysis is hypothesized to promote fibre cutting, greater fibrillation, and reduce refining energy costs. Despite potential benefits, there is little understanding of the mechanisms behind fibre development during enzymatic hydrolysis of mechanical pulp. This work investigates how incubation pH and temperature during enzymatic hydrolysis impact the refining of mechanical pulp short fibres. Incubation with endoglucanase at pH 5 and 60 °C increased fibre cutting by approximately 20 %. Fibrillation was negatively affected at this condition, resulting in increased slim fines formation with refining. Incubation at pH 8 and 80 °C promoted >15 % reduction in fibre length, despite such conditions being associated with low enzyme activity. The pH variation modified the sedimentation height of the fibres and the conductivity of suspensions, indicating a change in fibre surface charge. Fibre morphology changes were induced by enzyme hydrolysis conducted at conditions representative of the full range of pH and temperature observed in mechanical pulp mills.

Physicochemical, structural and functional properties of pomelo peel pectin extracted by combination of pulsed electric field and cellulase hydrolysis.

In this study, pectin extracted from pomelo peel was investigated using three different combination methods of pulsed electric field (PEF) and cellulase. Three action sequences were performed, including PEF treatment followed by enzymatic hydrolysis, enzymatic hydrolysis followed by PEF treatment, and enzymatic hydrolysis simultaneously treated by PEF. The three corresponding pectins were namely PEP, EPP and SP. The physiochemical, molecular structural and functional properties of the three pectins were determined. The results showed that PEP had excellent physiochemical properties, with the highest yield (12.08 %), total sugar (80.17 %) and total phenol content (38.20 %). The monosaccharide composition and FT-IR analysis indicated that the three pectins were similar. The molecular weights of PEP, EPP and SP were 51.13, 88.51 and 40.00 kDa, respectively. PEP showed the best gel properties, emulsification stability and antioxidant capacity among the three products, due to its high galacturonic acid and total phenol content, appropriate protein and low molecular weight. The mechanism of PEF-assisted cellulase hydrolysis of pomelo peel was also revealed by SEM analysis. These results suggested that PEF pretreatment was the best method, which not only improved the efficiency of enzymatic extraction, but also reduced resource waste and increased financial benefits.

Combinational manipulation of transcription factors, CreA and ClbR, is a viable strategy to improve cellulolytic enzyme production in Aspergillus aculeatus.

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, ß-1,4-xylanase, and ß-glucosidase production. Cellulase and ß-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. ß-Glucosidase production in ΔcreA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-ß-1,4-glucanase, ß-1,4-xylanase, and ß-glucosidase 1 (bgl1). Interestingly, bgl1 expression in ΔcreA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and ß-glucosidase production in A. aculeatus.

A sequential approach of alkali enzymatic extraction of dietary fiber from rice bran: Effects on structural, thermal, crystalline properties, and food application.

Rice bran is abundant in dietary fiber and is often referred to as the seventh nutrient, recognized for its numerous health benefits. The objective of the current study is to investigate the extraction of both soluble and insoluble dietary fiber from defatted rice bran (DRB) using an alkali-enzymatic treatment through response surface methodology. The independent variables like substrate percentage (5-30 %), enzyme concentration (1-50 µL/g), and treatment time (2-12 h) and dependent variables were the yield of soluble and insoluble DF. The highest extraction yield was observed with alkali enzyme concentration (50 µL/g) treatment, resulting in 2 % SDF and 59.5 % IDF at 24 h of extraction. The results indicate that cellulase-AC enzyme aids in the hydrolysis of higher polysaccharides, leading to structural alterations in DRB and an increase in DF yield. Furthermore, the disruption of intra-molecular hydrogen bonding between oligosaccharides and the starch matrix helps to increase in DF yield, was also confirmed through FTIR and SEM. The extracted DF soluble and insoluble was then used to develop rice porridge. Sensory evaluation using fuzzy logic analysis reported the highest scores for samples containing 0.5 % insoluble DF and 1.25 % soluble DF.

Effect of exogenous cellulase pretreatment of straw-type fermented total mixed ration on digestibility and ruminal fermentation in growing beef cattle.

Objective: This study aimed to assess the impact of corn straw-based unfermented and fermented total mixed rations (TMR) supplemented with exogenous cellulase on the in vitro fermentation characteristics, growth performance, feeding behavior, apparent digestibility, rumen fermentation and digestive enzyme activities of Chinese Simmental bulls. Methods: Unfermented (direct spraying of exogenous cellulase onto TMR, TMR) and fermented (exogenous cellulase fermentation for more than 7 d, fermented total mixed rations [FTMR]) TMR were collected, dried, powdered and used as fermentation substrates. The fermentation liquid was ruminal fluid collected from Chinese Simmental bulls. The artificial rumen culture fluid were continuously cultured in vitro for 48 h. Based on the diets they were fed, 24 healthy Chinese Simmental bulls (average weight of 495.93 ±10.89 kg) were randomly divided into two groups, with 12 bulls in each group, which were fed TMR or FTMR. The study lasted 56 d. Results: In in vitro experiments, the neutral detergent fiber degradability and total volatile fatty acid, propionate, iso-butyrate, iso-valerate and valerate concentrations were greater in the FTMR group (p<0.05) than in the TMR group. However, the methane production, pH and A/P of the FTMR group tended to be lower (p<0.05) than those of the TMR group. In the in vivo experiments, the average daily gain, eating rate, and feed efficiency of the FTMR groups were greater (p<0.05) than those of the TMR group. Similarly, the NDF degradability of the FTMR group was greater (p<0.05) than that of the TMR group. Compared to those in the TMR group, the concentrations of total volatile fatty acids, iso-butyrate, propionate and butyrate were greater in the FTMR group (p<0.05), and the A/P ratio was lower (p<0.05). Similarly, cellulase, xylanase, and ß-glucosidase activities were greater (p<0.05) in the FTMR group than in the TMR group. Conclusion: Corn straw-based fermented total mixed rations supplemented with exogenous cellulase play a vital role in decreasing the structural carbohydrate content of TMR and ruminal methane production in vitro, improving nutrient digestion and absorption, optimizing rumen fermentation, and improving the growth performance of beef cattle.

A modular enzyme with combined hemicellulose-removing and LPMO activity increases cellulose accessibility in softwood.

Because of the association with other complex polysaccharides, extracting and utilizing cellulose from lignocellulosic materials requires the combined action of a broad range of carbohydrate-active enzymes, including multiple glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The interplay between these enzymes and the way in which Nature orchestrates their co-existence and combined action are topics of great scientific and industrial interest. To gain more insight into these issues, we have studied the lignocellulose-degrading abilities of an enzyme from Caldibacillus cellulovorans (CcLPMO10-Man5), comprising an LPMO domain, a GH5 mannanase domain and two family 3 carbohydrate-binding modules (CBM3). Using a natural softwood substrate, we show that this enzyme promotes cellulase activity, i.e., saccharification of cellulose, both by removing mannan covering the cellulose and by oxidatively breaking up the cellulose structure. Synergy with CcLPMO10-Man5 was most pronounced for two tested cellobiohydrolases, whereas effects were smaller for a tested endoglucanase, which is in line with the notion that cellobiohydrolases and LPMOs attack the same crystalline regions of the cellulose, whereas endoglucanases attack semi-crystalline and amorphous regions. Importantly, the LPMO domain of CcLPMO10-Man5 is incapable of accessing the softwood cellulose in absence of the mannanase domain. Considering that LPMOs not bound to a substrate are sensitive to autocatalytic inactivation, this intramolecular synergy provides a perfect rationale for the evolution of modular enzymes such as CcLPMO10-Man5. The intramolecular coupling of the LPMO with a mannanase and two CBMs ensures that the LPMO is directed to areas where mannans are removed and cellulose thus becomes available.

A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation.

Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.

Review of research progress on the production of cellulase from filamentous fungi.

Cellulases have been widely used in many fields such as animal feed, textile, food, lignocellulose bioconversion, etc. Efficient and low-cost production of cellulases is very important for its industrial application, especially in bioconversion of lignocellulosic biomass. Filamentous fungi are currently widely used in industrial cellulase production due to their ability to secrete large amounts of active free cellulases extracellularly. This review comprehensively summarized the research progress on cellulases from filamentous fungi in recent years, including filamentous fungi used for cellulase production and its modification strategies, enzyme compositions, characterization methods and application of fungal cellulase systems, and the production of fungal cellulase includes production processes, factors affecting cellulase production such as inducers, fermentation medium, process parameters and their control strategies. Also, the future perspectives and research topics in fungal cellulase production are presented in the end of the review. The review helps to deepen the understanding of the current status of fungal cellulases, thereby promoting the production technology progress and industrial application of filamentous fungal cellulase.

Cellulase-responsive hydroxypropyl cellulose-anchored hollow mesoporous silica carriers for pesticide delivery.

In this study, a cellulase-responsive controlled-release formulation (FPR-HMS-HPC) was developed by grafting hydroxypropyl cellulose (HPC) onto fipronil (FPR) loaded hollow mesoporous silica (HMS) nanoparticles via ester linkage. The FPR-HMS-HPC formulation was characterized using scanning and transmission electron microscopies, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The results indicated that FPR-HMS-HPC exhibited a high loading capacity of 10.0 % (w/w) and demonstrated favorable responsiveness to cellulase enzyme. Moreover, its insecticidal efficacy against Reticulitermes flaviceps surpassed that of an equivalent dose of FPR. Toxicology studies showed that the mortality and hatching rates of zebrafish exposed to FPR-HMS-HPC nanoparticles were reduced by >6.5 and 8.0 times, respectively. Thus, HPC-anchored HMS nanoparticles as insecticide delivery systems present a sustainable method for pest control significantly reducing harm to non-target organisms and the environment.

Enhanced glucose-1-phosphate production from corn stover using cellulases with reduced ß-glucosidase activity via Trbgl1 gene knockout in Trichoderma reesei Rut C30.

The scarcity of cellulases with low ß-glucosidase activity poses a significant technological challenge in precisely controlling the partial hydrolysis of lignocellulose to cellobiose, crucial for producing high-value chemicals such as starch, inositol, and NMN. Trichoderma reesei is a primary strain in cellulase production. Therefore, this study targeted the critical ß-glucosidase gene, Trbgl1, resulting in over an 86 % reduction in ß-glucosidase activity. However, cellulase production decreased by 19.2 % and 20.3 % with lactose or cellulose inducers, respectively. Notably, transcript levels of cellulase genes and overall yield remained unaffected with an inducer containing sophorose. This indicates that ß-glucosidase BGL1 converts lactose or cellulose to sophorose through transglycosylation activity, inducing cellulase gene transcription. The resulting enzyme cocktail, comprising recombinant cellulase and cellobiose phosphorylase, was applied for corn stover hydrolysis, resulting in a 24.3 % increase in glucose-1-phosphate yield. These findings provide valuable insights into obtaining enzymes suitable for the high-value utilization of lignocellulose.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Screening and evaluation of the ruminal cellulolytic bacteria and their potential application as probiotics.

Background and Objectives: Rumen microbiologists are looking for new probiotics to improve the digestibility of livestock diets. This study intended to screen and evaluate the ruminal cellulolytic bacteria (CBs) and their potential application as probiotics. Materials and Methods: Microbial culture and molecular techniques performed to isolate CBs from the rumen of camels, deer and rams. Their antibacterial and antibiogram tests were done using disc diffusion method. Their potential to degrade cellulose, starch, tannin and protein were investigated using clear zone halo, and spectrophotometric techniques. Bilious, saline, and acidic broth media were used to study the resistance of isolates in intestinal conditions. Results: The phylogenetic analysis revealed that the strains belonged to Firmicutes and Proteobacteria phyla, Citrobacter murliniae, Ornithinibacillus bavariensis, C. braakii, and Bacillus subtilis. The highest cellulase (CAS) activity was recorded by C. murliniae Dez wildlife13A (2.98 UmL-1), whereas C. braakii Loot desert 111A (1.14 Uml-1) was produced the lowest enzyme. The isolates were highly resistant to synthetic conditions of intestine (pH 2.5-3.5, bile 0.3-2%), as well as tolerated higher concentrations of NaCl (up to 10%). They effectively inhibited standard pathogen strains, and showed sensitivity to the used antibiotics. Conclusion: This study reports the cellulolytic O. bavariensis Tabbas desert 32A for the first time from the rumen, which will have potential biotechnological applications.

Enzyme Activity of Culturable Fungi and Bacteria Isolated from Traditional Agarwood Fermentation Basin Indicate Temporally Significant Lignocellulosic and Lipid Substrate Modulations.

Agarwood oil is one of the costliest essential oils used in perfumery, medicine and aroma. Production of the oil traditionally involves a soaking/fermentation step. Studies have indicated a definite role of the diverse microorganisms growing during the open soaking step, and in the emergent aroma of the essential oil. However, the temporal nature of fermentation and a key functional aspect i.e., the enzymatic properties of the microbes from the fermentation basin have not been studied yet. A total of 20 bacteria and 14 fungi isolated from fermentation basins located in Assam, India, at different soaking periods classified as early (0-20 days), medium (20-40 days) and late (40-60 days) clearly pointed towards an early fungal domination followed by succession of bacteria. The physico-chemical transformations of the wood are controlled by enzymatic properties (cellulase, xylanase, amylase and lipase) of the isolates. The results indicated a strong lignocellulosic substrate modulation potential in the four isolates, viz- Purpureocillium lilacinum (0.354 mg/mL), Mucor circinelloides (0.331 mg/mL), Penicillium citrinum (0.324 mg/mL) and Bacillus megaterium (0.152 mg/mL). The highest culturable abundance (CFU/mL) was found in M. circinelloides (2 × 109) among fungi and B. megaterium (4.5 × 109) among bacteria. The highest cellulase activity was shown by P. lilacinum (0.354 mg/mL) while xylanase and lipase by M. circinelloides (0.873 and 0.128 mg/mL). An interesting revelation was that a substantial proportion of the isolates (70% bacteria and 78% fungi) were positive for lipase activity. This is the first report on the "culturable microbiome" of the agarwood fermentation basin from a temporal and functional bioactivity perspective. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01257-y.

Screening the Rumen of Balochi Camel (Camelus dromedarius) and Cashmere Goat (Capra hircus) to Isolate Enzyme-Producing Bacteria as Potential Additives for Animal Feed.

Rumen microbiology has made a significant contribution to the discovery of biodegradation processes, which convert nutrients into energy for ruminants. Therefore, understanding the enzymatic potential in the rumen of different animal species is essential for developing efficient microbial feed additives. The aim of this study was to isolate enzyme-producing bacteria (EPBs) from the rumen of the Balochi camel (Camelus dromedarius) and Cashmere goat (Capra hircus) as potential additives for animal feed. The EPBs were screened based on the hydrolysis of carboxyl methyl cellulose, tannin, starch, and bovine serum albumin. The isolates were then subjected to enzyme activity assays and molecular characterization. Additionally, they were evaluated for their antagonistic effects, antibiotic susceptibility, and growth in acidic, bile, and saline media. Thirteen enzyme-producing strains were identified in the rumen of the camels and goats, belonging to the genera Klebsiella, Escherichia, Raoultella, Enterobacter and Pectobacterium. The highest and lowest tannase activities were recorded for Escherichia coli GHMGHE41 (10.46 Um/l-1) and Raoultella planticola GHMGHE15 (1.83 Um/l-1), respectively. Enterobacter cloacae GHMGHE18 (2.03 U/ml) was the most effective cellulolytic isolate, compared to Klebsiella strains (1.05 Um/l-1). The highest protease producer was Klebsiella pneumoniae GHMGHE13 (3.00 U/ml-1), while Escherichia coli GHMGHE17 (1.13 U/ml-1) had the lowest activity. Klebsiella pneumoniae GHMGHE13 (1.55 U/ml-1) and Enterobacter cloacae GHMGHE19 (1.26 U/ml-1) were the highest and lowest producers of amylase, respectively. The strains exhibited mixed responses to antibiotics and remained stable under stressful conditions. These findings indicate that ruminal EPBs have the potential to be used in animal feed, pending further in vivo studies.

Structural insights and functional characterization of a novel ß-glucosidase derived from Thermotoga profunda.

ß-Glucosidase is a crucial cellulase, as its activity determines the efficiency of cellulose hydrolysis into glucose. This study addresses the functional and structural characteristics of Thermotoga profunda ß-glucosidase (Tp-BGL). Tp-BGL exhibited a Km of 0.3798 mM for p-nitrophenyl-ß-d-glucopyranoside (pNPGlc) and 4.44 mM for cellobiose, with kcat/Km of 1211.16 and 4.18 s-1 mM-1, respectively. In addition, Tp-BGL showed significant pH adaptability and thermal stability, with a Tm of 85.7 °C and retaining >90 % of its activity after incubation at 80 °C for 90 min. The crystal structure of Tp-BGL was resolved at 1.95 Å resolution, and reveals a typical TIM barrel structure. Comparative structural analysis highlighted that the major distinction between Tp-BGL and the other glucosidases lies in their loop regions.

Enzyme immobilization with nanomaterials for hydrolysis of lignocellulosic biomass: Challenges and future Perspectives.

Enzyme immobilization has emerged as a prodigious strategy in the enzymatic hydrolysis of lignocellulosic biomass (LCB) promising enhanced efficacy and stability of the enzymes. Further, enzyme immobilization on magnetic nanoparticles (MNPs) facilitates the easy recovery and reuse of biocatalysts. This results in the development of a nanobiocatalytic system, that serves as an eco-friendly and inexpensive LCB deconstruction approach. This review provides an overview of nanomaterials used for immobilization with special emphasis on the nanomaterial-enzyme interactions and strategies of immobilization. After the succinct outline of the immobilization procedures and supporting materials, a comprehensive assessment of the catalysis enabled by nanomaterial-immobilized biocatalysts for the conversion and degradation of lignocellulosic biomasses is provided by gathering state-of-the-art examples. The challenges and future directions associated with this technique providing a potential solution in the present article. Insight on the recent advancements in the process of nanomaterial-based immobilization for the hydrolysis of lignocellulosic biomass has also been highlighted in the article.

Soil-derived cellulose-degrading bacteria: screening, identification, the optimization of fermentation conditions, and their whole genome sequencing.

Straw cellulose is an abundant renewable resource in nature. In recent years, the conversion of cellulose from waste straw into biofuel by specific microorganisms' fragmentation has attracted extensive attention. Although many bacteria with the ability to degrade cellulose have been identified, comprehensive bioinformatics analyses of these bacteria remain limited, and research exploring optimal fragmentation conditions is scarce. Our study involved the isolation and screening of bacteria from various locations in Yangzhou using carboxymethyl cellulose (CMC) media. Then, the cellulose-degrading bacteria were identified using 16S rRNA and seven candidate bacterial strains with cellulose degrading ability were identified in Yangzhou city for the first time. The cellulase activity was determined by the 3,5-dinitrosalicylic acid (DNS) method in different fragmentation conditions, and finally two bacteria strains with the strongest cellulose degradation ability were selected for whole genome sequencing analysis. Sequencing results revealed that the genome sizes of Rhodococcus wratislaviensis YZ02 and Pseudomonas Xanthosomatis YZ03 were 8.51 Mb and 6.66 Mb, containing 8,466 and 5,745 genes, respectively. A large number of cellulose degradation-related genes were identified and annotated using KEGG, GO and COG analyses. In addition, genomic CAZyme analysis indicated that both R. wratislaviensis YZ02 and P. Xanthosomatis YZ03 harbor a series of glycoside hydrolase family (GH) genes and other genes related to cellulose degradation. Our finding provides new options for the development of cellulose-degrading bacteria and a theoretical basis for improving the cellulose utilization of straw.

Comparison of simultaneous saccharification and fermentation with LPMO-supported hybrid hydrolysis and fermentation.

Enzymatic saccharification is used to convert polysaccharides in lignocellulosic biomass to sugars which are then converted to ethanol or other bio-based fermentation products. The efficacy of commercial cellulase preparations can potentially increase if lytic polysaccharide monooxygenase (LPMO) is included. However, as LPMO requires both a reductant and an oxidant, such as molecular oxygen, a reevaluation of process configurations and conditions is warranted. Saccharification and fermentation of pretreated softwood was investigated in demonstration-scale experiments with 10 m3 bioreactors using an LPMO-containing cellulase preparation, a xylose-utilizing yeast, and either simultaneous saccharification and fermentation (SSF) or hybrid hydrolysis and fermentation (HHF) with a 24-hour or 48-hour initial phase and with 0.15 vvm aeration before addition of the yeast. The conditions used for HHF, especially with 48 h initial phase, resulted in better glucan conversion, but in poorer ethanol productivity and in poorer initial ethanol yield on consumed sugars than the SSF. In the SSF, hexose sugars such as glucose and mannose were consumed faster than xylose, but, in the end of the fermentation >90% of the xylose had been consumed. Chemical analysis of inhibitory pretreatment by-products indicated that the concentrations of heteroaromatic aldehydes (such as furfural), aromatic aldehydes, and an aromatic ketone decreased as a consequence of the aeration. This was attributed mainly to evaporation caused by the gas flow. The results indicate that further research is needed to fully exploit the advantages of LPMO without compromising fermentation conditions.

Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyceslactis.

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.