Antibiotic-free production of sucrose isomerase in Bacillus subtilis by genome integration.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose to form isomaltulose, a valuable functional sugar widely used in the food industry. However, the lack of safe and efficient heterologous expression systems hinders SIase production and application. In this study, we achieved antibiotic-free SIase expression in Bacillus subtilis through genome integration. Using CRISPR/Cas9 system, SIase expression cassettes were integrated into various genomic loci, including amyE and ctc, both individually and in combination, resulting in single-copy and muti-copy integration strains. Engineered strains with a maltose-inducible promoter effectively expressed and secreted SIase. Notably, multi-copy strain exhibited enhanced SIase production, achieving 4.4 U/mL extracellular activity in shake flask cultivations. Furthermore, crude enzyme solution from engineered strain transformed high concentrations sucrose into high yields of isomaltulose, reaching a maximum yield of 94.6%. These findings demonstrate antibiotic-free SIase production in B. subtilis via genome integration, laying the foundation for its industrial production and application.

Silica-magnesium coupling in lignin-based biochar: A promising remediation for composite heavy metal pollution in environment.

Lignin hydrothermal silica-carbon material served as a backbone for MgCl2 activation to prepare lignin-based silicon/magnesia biochar (ALB/Si-Mg) for Cd2+, Pb2+, Cu2+, and Zn2+ removal from water and soil environment. Characterization studies revealed a 1017.71-fold increase in the specific surface area of ALB/Si-Mg compared to the original lignin biochar (ALB), producing abundant oxygen functional groups (OC-O, Si-O, Mg-O), and mineral matter (Mg2SiO4 and MgO). Crucially, batch adsorption experiments demonstrated that the adsorption capacity of ALB/Si-Mg for Cd2+, Pb2+, Cu2+, and Zn2+ was 848.17, 665.07, 151.84, and 245.78 mg/g, which were 29.09-140.45 times of the ALB. Soil remediation experiments showed that applying ALB/Si-Mg increased soil effective silicon (109.04%-450.2%) and soil exchangeable magnesium (276.41%-878.66%), enhanced plant photosynthesis, and notably reduced the bioavailability of heavy metals in soil as well as the content of heavy metals in Pakchoi, thereby promoting Pakchoi growth and development. The presence of oxygen-containing functional groups on ALB/Si-Mg, along with Mg2SiO4 and MgO nanoparticles, enhanced the adsorption capacity for heavy metals through the promotion of heavy metal precipitation, ion exchange, and complexation mechanisms. This study establishes the groundwork for the coupling of silica and magnesium elements in biochar and the remediation of composite heavy metal environmental pollution.

Development of an engineered Bacillus subtilis strain for antibiotic-free sucrose isomerase production.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.

Pretreatment of chitin depolymerization by expansin-like protein to improve hydrolysis efficiency.

Chitin is a polysaccharide similar to cellulose that contains abundant hydrogen bonds. Expansin-like proteins disrupt hydrogen bond networks, causing cellulose to swell and accelerating its degradation. We examined the effects of pretreatment with two expansin-like proteins, CxEXL22 (Arthrobotrys sp. CX1) and HcEXL (Hahella chejuensis), on chitin depolymerisation and enzymatic degradation. The efficiency of chitin degradation increased more than two-fold after pretreatment with expansin-like proteins. Following pretreatment with expansin-like proteins, chitin had a lower crystallinity index, greater d-spacing and crystallite size, and weaker hydrogen bonds, and the loosened porous microfibrils were more exposed than in untreated chitin. The rupture characterisation of crystalline chitin indicated that expansin-like proteins loosened the hydrogen bonds of the chitin polysaccharide chains, causing significant depolymerisation to expose more porous structures and enhance chitin accessibility.

High-Level Expression and Biochemical Characterization of a Maltotetraose Amylase in Pichia pastoris X-33 for Maltotetraose Production.

Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1 g/L, and its kcat was 3237.6 s-1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200 g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application.

Environmental applications of lignin-based hydrogels for Cu remediation in water and soil: adsorption mechanisms and passivation effects.

Heavy metal pollution, particularly the excessive release of copper (Cu), is an urgent environmental concern. In this study, sodium lignosulfonate/carboxymethyl sa-son seed gum (SL-Cg-g-PAA) designed for remediation of Cu-contaminated water and soil was successfully synthesized through a free radical polymerization method using lignin as a raw material. This hydrogel exhibits remarkable Cu adsorption capability when applied to water, with a maximum adsorption capacity reaching 172.41 mg/g. Important adsorption mechanisms include surface complexation and electrostatic attraction between Cu(Ⅱ) and oxygen-containing functional groups (-OH, -COOH), as well as cation exchange involving -COONa and -SO3Na. Furthermore, SL/Cg-g-PAA effectively mitigated the bioavailability of heavy metals within soil matrices, as evidenced by a notable 14.1% reduction in DTPA extracted state Cu (DTPA-Cu) content in the S4 treatment (0.7% SL/Cg-g-PAA) compared to the control group. Concurrently, the Cu content in both the leaves and roots of pakchoi exhibited substantial decreases of 55.19% and 36.49%, respectively. These effects can be attributed to the precipitation and complexation reactions facilitated by the hydrogel. In summary, this composite hydrogel is highly promising for effective remediation of heavy metal pollution in water and soil, with a particular capability for the immobilization of Cu(Ⅱ) and reduction of its adverse effects on ecosystems.

Glucose Starvation Stimulates the Promoting Strength of a Novel Evolved Suc2 Promoter.

Promoters are essential for designing Saccharomyces cerevisiae cell factories. Identifying novel promoters tuned to produce specific metabolites under increasingly diverse industrial stresses is required to improve the economic feasibility of whole fermentation processes. In this study, a positively evolved Suc2 promoter (SUC 2p) with promoter activity stronger than that of the wild-type Suc2 promoter (SUC 2wtp) was obtained. Quantitative real-time PCR, fluorescence analysis, Western blotting, and a ß-galactosidase activity assay revealed that SUC 2p is a medium-strength promoter compared with eight reported promoters at a medium glucose concentration (2% (w/v)). Different glucose concentrations modulated the strength of SUC 2p. Low glucose concentrations (0.05 and 0.5% (w/v)) enhanced the promoter strength of SUC 2p dramatically, with promoter activity higher than that of reported strong promoters. Glucose starvation resulted in the formation of a new Msn2/4 binding site on SUC 2p. Our work should facilitate the development of promoters with novel fine-tuning properties and the construction of S. cerevisiae cell factories suitable for the industrial production of essential chemicals under glucose-deprived conditions.

An aliphatic MOF with a molecular sieving effect for efficient C2H2/C2H4 separation.

A metal-organic framework, SDMOF-1, with rigid pores of about 3.4 Å, which is appropriate for accommodating C2H2 molecules, exhibits high C2H2 adsorption capacity and great separation capability of the C2H2/C2H4 mixture. This work provides a new method to design aliphatic MOFs with a molecular sieving effect to realize efficient gas separation.

Degradation of oxytetracycline by iron-manganese modified industrial lignin-based biochar activated peroxy-disulfate: Pathway and mechanistic analysis.

In this study, high-performance Fe-Mn-modified industrial lignin-based biochar (FMBC) was successfully prepared to facilitate the efficient degradation of oxytetracycline by its driven sulfate radical-based advanced oxidation process with 90% degradation within 30 min. The results showed that oxygenated functional groups (e. g. hydroxyl, carbonyl, etc.) in industrial lignin-based biochar, the synergistic effect of transition metals Fe and Mn, and defective structures were the active sites for activation of peroxy-disulfate. SO4·- produced during the degradation process assumed a key function. Significantly, 38 intermediates were innovatively proposed for the first time in the system, and oxytetracycline was degraded in 7 ways, including deamidation, demethylation, hydroxylation, secondary alcohol oxidation, ring opening, dehydration, and carbonylation. A new perspective on the application of industrial lignin in the advanced oxidative degradation of organic pollutants was provided by this study.

Expression and biochemical characterization of a Bacillus subtilis catalase in Pichia pastoris X-33.

Catalase, which catalyzes the decomposition of H2O2 to H2O and O2, is widely used to reduce H2O2 in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 ± 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 °C and pH 11.0. Its Km for hydrogen peroxide was 10.9 ± 0.5 mM, and its kcat/Km was 5788.1 ± 25.6 s-1 mM-1. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.

An Insight into the Essential Role of Carbohydrate-Binding Modules in Enzymolysis of Xanthan.

To date, due to the low accessibility of enzymes to xanthan substrates, the enzymolysis of xanthan remains deficient, which hinders the industrial production of functional oligoxanthan. To enhance the enzymatic affinity against xanthan, the essential role of two carbohydrate binding modules-MiCBMx and PspCBM84, respectively, derived from Microbacterium sp. XT11 and Paenibacillus sp. 62047-in catalytic properties of endotype xanthanase MiXen were investigated for the first time. Basic characterizations and kinetic parameters of different recombinants revealed that, compared with MiCBMx, PspCBM84 dramatically increased the thermostability of endotype xanthanase, and endowed the enzyme with higher substrate affinity and catalytic efficiency. Notably, the activity of endotype xanthanase was increased by 16 times after being fused with PspCBM84. In addition, the presence of both CBMs obviously enabled endotype xanthanase to produce more oligoxanthan, and xanthan digests prepared by MiXen-CBM84 showed better antioxidant activity due to the higher content of active oligosaccharides. The results of this work lay a foundation for the rational design of endotype xanthanase and the industrial production of oligoxanthan in the future.

High capacity adsorption of oxytetracycline by lignin-based carbon with mesoporous structure: Adsorption behavior and mechanism.

In this study, an adsorbent with mesoporous structure and PO/PO bonds is prepared by hydrothermal and phosphoric acid activation from industrial alkali lignin for the adsorption of oxytetracycline (OTC). The adsorption capacity is 598 mg/g, which is three times higher than that of the adsorbent with microporous structure. The rich mesoporous structure of the adsorbent provides adsorption channels and filling sites, and π-π attraction, cation-π interaction, hydrogen bonds, and electrostatic attraction provide adsorption forces at the adsorption sites. The removal rate of OTC exceeds 98 % over a wide range of pH values (3-10). It has high selectivity for competing cations in water, with higher than 86.7 % removal rate of OTC from medical wastewater. After 7 consecutive adsorption-desorption cycles, the removal rate of OTC remains as high as 91 %. This efficient removal rate and excellent reusability indicate the strong potential of the adsorbent for industrial applications. This study prepares a highly efficient, environmentally friendly antibiotic adsorbent that can not only efficiently remove antibiotics from water but also recycle industrial alkali lignin waste.

Removal of Cd2+ from wastewater to form a three-dimensional fiber network using Si-Mg doped industrial lignin-based carbon materials.

In this study, SiMg doped industrial lignin-based carbon materials (SLCs) were prepared by water bath silicification and MgCl2 activation to remove Cd2+ from aqueous solutions. What's more, the doping of SiMg jointly promoted the excellent physicochemical properties of the material, e.g., high specific surface area, good pore volume, and numerous oxygen-containing groups. The Cd2+ batch adsorption experiments proved that SLCs have good Cd2+ removal capacity within pH 3-7, and the adsorption model demonstrated the adsorption process as a physicochemically complex process. The maximum adsorption of Cd2+ in the SLC was 665.35 mg/g, and the contributing factors to the removal of Cd2+ were as follows: ion exchange (59.36 %) > Cd2+ precipitation (24.93 %) > oxygen-containing functional group complexation (14.79 %) > Cd2+-π interactions (0.92 %). In addition, the complexation of SiO, MgO, and Cd precipitates allowed the formation of a three-dimensional fiber mesh structure. The application of SLCs has the potential to eliminate Cd2+ pollution in water bodies, and its preparation is simple and environmentally friendly. Finally, this study provides a theoretical basis for an in-depth understanding of the mechanism of heavy metal adsorption by inorganic nonmetals in combination with metal oxides.

Inhibition of Chitosan with Different Molecular Weights on Barley-Borne Fusarium graminearum during Barley Malting Process for Improving Malt Quality.

There are many Fusarium graminearum contaminations in barley that are often associated with malt and beer quality issues. Thus, it is important to find a biological antifungal agent to prevent the growth of F. graminearum during malting. Minimum inhibition concentration (MIC) of chitosan for mycelial growth and spore germination of F. graminearum was 2.6 g/L and 1.6 g/L, respectively, indicating that the F. graminearum strain was highly sensitive toward chitosan. Chitosan with a molecular weight of 102.7 kDa was added at 0.5 g/kg during the first steeping stage, resulting in the maximum inhibition rate of F. graminearm in barley. The biomass of F. graminearm and deoxynivalenol content in the infected barley at the end of germination with 0.5 g/kg chitosan treatment were decreased by 50.7% and 70.5%, respectively, when compared with the infected barley without chitosan. Chitosan could remove the negative effects of F. graminearm infection on barley germination and malt quality, which makes the application of chitosan during the steeping process as a potential antifungal agent in the malting process to protect from F. graminearum infection.

Enhanced Extracellular Production and Characterization of Sucrose Isomerase in Bacillus subtilis with Optimized Signal Peptides.

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, which is an important functional sugar widely used in the food industry. However, the lack of safe and efficient expression systems for recombinant SIase has impeded its production and application. In this study, enhanced expression of a SIase from Klebsiella sp. LX3 (referred to as KsLX3-SIase) was achieved in Bacillus subtilis WB800N, by optimizing the signal peptides. First, 13 candidate signal peptides were selected using a semi-rational approach, and their effects on KsLX3-SIase secretion were compared. The signal peptide WapA was most efficient in directing the secretion of KsLX3-SIase into the culture medium, producing a specific activity of 23.0 U/mL, as demonstrated by shake flask culture. Using a fed-batch strategy, the activity of KsLX3-SIase in the culture medium was increased to 125.0 U/mL in a 5-L fermentor. Finally, the expressed KsLX3-SIase was purified and was found to have maximum activity at 45 °C and pH 5.5. Its Km for sucrose was 267.6 ± 18.6 mmol/L, and its kcat/Km was 10.1 ± 0.2 s-1mM-1. These findings demonstrated an efficient expression of SIase in B. subtilis, and this is thought to be the highest level of SIase produced in a food-grade bacteria to date.

Novel ß-Glucosidase Mibgl3 from Microbacterium sp. XT11 with Oligoxanthan-Hydrolyzing Activity.

The enzymatic pathway of xanthan depolymerization has been predicted previously; however, the ß-glucosidase and unsaturated glucuronyl hydrolase in this system have not been cloned and characterized. This lack of knowledge hinders rational modification of xanthan and exploration of new applications. In this work, we report on the properties of Mibgl3, a xanthan-degrading enzyme isolated from Microbacterium sp. XT11. Mibgl3 exhibits typical structural features of the GH3 family but shares low sequence identity with reported GH3 enzymes. The activity of Mibgl3 can be inhibited by Cu2+, Fe2+, Zn2+, and glucose. Unlike most ß-glucosidases, Mibgl3 can tolerate a wide pH range and is activated by high concentrations of NaCl. This improves the commercial value of Mibgl3. In particular, Mibgl3 exhibits higher substrate specificity toward oligoxanthan than other ß-glucosidases. Ion chromatography, ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-MS), and GC-MS results showed that Mibgl3 could effectively hydrolyze oligoxanthan to release glucose and glucuronate. Therefore, Mibgl3 might play an important role in xanthan depolymerization by functioning as hydrolase of both the xanthan backbone and sidechains. This knowledge of the enzymatic properties and hydrolysis mechanism of a ß-glucosidase will be beneficial for future applications.

A comprehensive study of the differences in protein expression and chemical constituents in tea leaves (Camellia sinensis var. sinensis) with different maturity using a combined proteomics and metabolomics method.

The maturity of tea leaves has a great influence on the flavor quality and commercial price of tea. In this work, a combined proteomics and metabolomics analysis was applied to investigate the differences in protein expression and metabolites among tea leaves with different maturity. Integrated analysis showed that there were significant differences in 112 nonvolatile components related to the pathways of photosynthesis, glycolysis, tricarboxylic acid cycle, and the biosynthesis of amino acids, phenylpropanoids and flavonoids. The bud had higher expression levels of most enzymes related to the biosynthesis of amino acids, phenylpropanoids, and flavonoids, leading to higher levels of amino acids, most flavanols, and procyanidins compared with the leaves. The 1st leaf showed a higher expression level of flavonol synthase, which produces higher levels of flavonol-3-glycosides. This study offers deep insight into the maturity of tea at both the protein and metabolite levels and provides a guideline for tea manufacturing.

Mechanism of a double-channel nitrogen-doped lignin-based carbon on the highly selective removal of tetracycline from water.

A high-performance nitrogen-doped lignin-based carbon material (ILAC-N) was synthesized using industrial lignin and urea by hydrothermal and activation, as an absorbent of tetracycline hydrochloride (TC). The results showed that the ILAC-N comprises a double-channeled structure with micro and mesopores. It exhibits an excellent adsorption capacity of TC across a wide pH range (pH 2-11), with the highest adsorption capacity of 1396 mg g-1 at 323 K. Tests in actual wastewater showed that the TC removal rate by ILAC-N exceeded 97.4%. Moreover, it maintained a removal rate of 84% after 10 regeneration cycles, revealing its high reusability. Mechanisms suggested that pore filling and π-π interaction played a critical role in this process. In conclusion, ILAC-N can be broadly applied to livestock manure and pharmaceutical wastewater treatment, owing to its high adsorption capacity, good adsorption properties across a wide pH range, excellent reusability. Furthermore, this research opens a new path for lignin utilization.

[Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers].

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.

A novel accessory protein ArCel5 from cellulose-gelatinizing fungus Arthrobotrys sp. CX1.

Improved understanding of cellulose swelling mechanism is beneficial for increasing the hydrolysis efficiency of cellulosic substrates. Here, we report a family 5 glycoside hydrolase ArCel5 isolated from the cellulose-gelatinizing fungus Arthrobotrys sp. CX1. ArCel5 exhibited low specific hydrolysis activity and high cellulose swelling capability, which suggested that this protein might function as an accessory protein. Homology modeling glycosylation detection revealed that ArCel5 is a multi-domain protein including a family 1 carbohydrate-binding module, a glycosylation linker, and a catalytic domain. The adsorption capacity, structural changes and hydrature index of filter paper treated by different ArCel5 mutants demonstrated that CBM1 and linker played an essential role in recognizing, binding and decrystallizing cellulosic substrates, which further encouraged the synergistic action between ArCel5 and cellulases. Notably, glycosylation modification further strengthened the function of the linker region. Overall, our study provides insight into the cellulose decrystallization mechanism by a novel accessory protein ArCel5 that will benefit future applications.