An enzymatic hydrolysis-based platform technology for the efficient high-yield production of cellulose nanospheres.

This study evaluates the feasibility of using enzymatic technology to produce novel nanostructures of cellulose nanomaterials, specifically cellulose nanospheres (CNS), through enzymatic hydrolysis with endoglucanase and xylanase of pre-treated cellulose fibers. A statistical experimental design facilitated a comprehensive understanding of the process parameters, which enabled high yields of up to 82.7 %, while maintaining a uniform diameter of 54 nm and slightly improved crystallinity and thermal stability. Atomic force microscopy analyses revealed a distinct CNS formation mechanism, where initial fragmentation of rod-like nanoparticles and subsequent self-assembly of shorter rod-shaped nanoparticles led to CNS formation. Additionally, adjustments in process parameters allowed precise control over the CNS diameter, ranging from 20 to 100 nm, highlighting the potential for customization in high-performance applications. Furthermore, this study demonstrates how the process framework, originally developed for cellulose nanocrystals (CNC) production, was successfully adapted and optimized for CNS production, ensuring scalability and efficiency. In conclusion, this study emphasizes the versatility and efficiency of the enzyme-based platform for producing high-quality CNS, providing valuable insights into energy consumption for large-scale economic and environmental assessments.

Biodegradation mechanism of chlorpyrifos by halophilic bacterium Hortaea sp. B15.

For decades, most of the developing nations have relied on chlorpyrifos for insecticidal activity in the agriculture sector. It is a common chlorinated organophosphorus pesticide that has been widely used to control insects to protect plants. This study aimed to investigate the effects of environmental characteristics such as salinity, pH, temperature, and surfactant on Hortaea sp. B15 mediated degradation of chlorpyrifos as well as enzyme activity and metabolic pathway. The highest bacterial growth (4.6 × 1016 CFU/mL) was achieved after 20 h of incubation in a 100 mg/L chlorpyrifos amended culture. The fit model and feasible way to express the chlorpyrifos biodegradation kinetics in normal condition and optimized was a first-order rate equation, with an R2 value of 0.95-0.98. The optimum pH for chlorpyrifos biodegradation was pH 9, which resulted in a high removal rate (91.1%) and a maximum total count of 3.8 × 1016 CFU/mL. Increasing the temperature over 40 °C may inhibit microbial development and biodegradation. There was no significant effect of culture salinity on degradation and bacterial growth. In the presence of non-ionic surfactant Tween 80, the maximum chlorpyrifos degradation (89.5%) and bacterial growth (3.8 × 1016 CFU/mL) was achieved. Metabolites such as 3,5,6-trichloropyridin-2-ol and 2-pyridinol were identified in the Hortaea sp. B15 mediated degradation of chlorpyrifos. According to the findings, Hortaea sp. B15 should be recommended for use in the investigation of in situ biodegradation of pesticides.

Enzymatic and chemo-enzymatic strategies to produce highly valuable chiral amines from biomass with ω-transaminases on 2D zeolites.

Amino transaminases (ATAs) have been supported on a 2D ITQ-2 zeolite through electrostatic interactions, resulting in a highly stable active biocatalyst to obtain a variety of valuable chiral amines starting from prochiral ketones derived from biomass. We have extended the biocatalyst applications by designing a chemo-enzymatic process that allows, as the first step, prochiral ketones to be obtained from biomass-derived compounds through an aldol condensation-reduction step using a bifunctional metal/base catalyst. The prochiral ketone is subsequently converted into the chiral amine using the immobilized ATA. We show that it is feasible to couple both steps in a semi-continuous process to produce industrially relevant chiral amines with yields of >95% and ∼100% enantiomer excess.

Biological Soil Crusts as Ecosystem Engineers in Antarctic Ecosystem.

Biological soil crusts (BSC) are considered as pivotal ecological elements among different ecosystems of the world. The effects of these BSC at the micro-site scale have been related to the development of diverse plant species that, otherwise, might be strongly limited by the harsh abiotic conditions found in environments with low water availability. Here, we describe for the first time the bacterial composition of BSCs found in the proximities of Admiralty Bay (Maritime Antarctica) through 16S metabarcoding. In addition, we evaluated their effect on soils (nutrient levels, enzymatic activity, and water retention), and on the fitness and performance of Colobanthus quitensis, one of the two native Antarctic vascular plants. This was achieved by comparing the photochemical performance, foliar nutrient, biomass, and reproductive investment between C. quitensis plants growing with or without the influence of BSC. Our results revealed a high diversity of prokaryotes present in these soil communities, although we found differences in terms of their abundances. We also found that the presence of BSCs is linked to a significant increase in soils' water retention, nutrient levels, and enzymatic activity when comparing with control soils (without BSCs). In the case of C. quitensis, we found that measured ecophysiological performance parameters were significantly higher on plants growing in association with BSCs. Taken together, our results suggest that BSCs in Antarctic soils are playing a key role in various biochemical processes involved in soil development, while also having a positive effect on the accompanying vascular flora. Therefore, BSCs would be effectively acting as ecosystem engineers for the terrestrial Antarctic ecosystem.

A unique biochemical reaction pathway towards trehalulose synthesis by an amylosucrase isolated from Deinococcus deserti.

The aim of this study was to establish an efficient bioprocess for the synthesis of trehalulose as a novel sweetener. This disaccharide has 70% of the sweetness of sucrose and bioactive properties such as anti-cariogenicity and anti-oxidizing activity. In this study, amylosucrase from the Deinococcus deserti (DdAS) gene was expressed and purified. When DdAS was reacted with 2 M sucrose at 35 °C for 120 h, the yield ratio of trehalulose to turanose was approximately 2:1. The trehalulose yield increased when extrinsic fructose was added. Under optimum conditions for trehalulose synthesis, the yield reached 36% (246 g/L, sucrose basis) starting with 2 M sucrose + 0.75 M fructose and showed the highest trehalulose productivity (1.94 g/L/h). As a result, a novel amylosucrase that synthesized trehalulose as the major product was developed, in contrast to other studied amylosucrase-type enzymes. DdAS could be utilized industrially in a bioprocess for producing trehalulose as a functional sucrose alternative.

Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution.

Cytochrome P450 enzymes have attracted much interest over the years given their ability to insert oxygen into saturated carbon-hydrogen bonds, a difficult feat to accomplish by traditional chemistry. Much of the activity in this field has centered on the bacterial enzyme CYP102A1, or BM3, from Bacillus megaterium, as it has shown itself capable of hydroxylating/acting upon a wide range of substrates, thereby producing industrially relevant pharmaceuticals, fine chemicals, and hormones. In addition, unlike most cytochromes, BM3 is both soluble and fused to its natural redox partner, thus facilitating its use. The industrial use of BM3 is however stifled by its instability and its requirement for the expensive NADPH cofactor. In this work, we added several mutations to the BM3 mutant R966D/W1046S that enhanced the turnover number achievable with the inexpensive cofactors NADH and NBAH. These new mutations, A769S, S847G, S850R, E852P, and V978L, are localized on the reductase domain of BM3 thus leaving the oxidase domain intact. For NBAH-driven reactions by new mutant NTD5, this led to a 5.24-fold increase in total product output when compared to the BM3 mutant R966D/W1046S. For reactions driven by NADH by new mutant NTD6, this enhanced total product output by as much as 2.3-fold when compared to the BM3 mutant R966D/W1046S. We also demonstrated that reactions driven by NADH with the NTD6 mutant not only surpassed total product output achievable by wild-type BM3 with NADPH but also retained the ability to use this latter cofactor with greater total product output as well.

Recent advancements in the synthesis of novel thermostable biocatalysts and their applications in commercially important chemoenzymatic conversion processes.

Thermostable enzymes are a field of growing interest in bioremediation, pharmaceuticals, food industry etc., due to their ability to catalyze bio reactions at high temperatures. This review aims to provide an overview on extremophiles with a special focus on thermophiles and enzymes produced from extremophilic bacteria. Novel thermostable catalysts, used in producing commercially important chemicals, are discussed in this review. Various classes of enzymes produced by microbes, synthesis of thermozymes and comparison with enzymes produced at optimal conditions are critically discussed. A detailed discussion on immobilized enzymes in comparisons with free enzymes, produced by extremozymes, is included. Different parameters which affect enzyme production are also discussed. The current industrial trends along with the future of biocatalysts in the production of chemicals using efficient methods are also discussed.

Potential of enzymatic process as an innovative technology to remove anticancer drugs in wastewater.

Anticancer drugs are a class of pharmaceutical compounds that have been found in hospital, domestic, and industrial wastewaters and also in surface waters. They have been showing recalcitrance to conventional wastewater treatment technologies and present a potential risk to environment and human health, since they exhibit cytotoxic, teratogenic, and carcinogenic among other effects in higher organisms, even at low concentrations. The presence of these compounds in the environment is a recent challenge for wastewater treatment and some alternative strategies to remove them were already studied, such as white-rot fungi (WRF) technologies. Despite promising results, processes involving fungi are complex, have high reaction times, and require nutrient addition for fungus growth and maintenance. Due to this potential, strategies to make the technology feasible were studied, such as the possibility for direct application of enzymes secreted by WRF. Enzymatic processes were studied in the removal of other pharmaceuticals such as antibiotics, anti-inflammatory, and steroid hormones; however, to the best of our knowledge, there is a gap on literature about their direct action on anticancer drugs.

Alternative Process for Production of Sweet Potato Distilled Beverage

Abstract Shochu is the most widely consumed spirit in Japan. In its manufacture is used koji, a solid fungus culture traditional of the Asian countries, but that makes the production process slow. Shochu can be produced from a variety of starchy sources, including sweet potato. About 7% of the world's sweet potato production is wasted due to imperfections that make it unsuitable for consumption. However, this material can be used in ethanol production. Considering the high productivity of sweet potato in Brazil, an opportunity to add value to this raw material is perceived. An alternative process for the production of sweet potato distillate similar to shochu was proposed. Koji was replaced by a mixture of alpha-amylase and glucoamylase. Process time was reduced from 14 to only 1 day. Composition analyses were performed by HPLC and GC. The experimental yield of alcoholic fermentation using pectinase enzyme reached 67.31-73.65%, but methanol was above the limits of the legislation. Without the addition of pectinase, no methanol was formed. However, there was a decrease in yield (51.65-54.75%), due to the incomplete disintegration of sweet potatoes. The distillate produced and the commercial shochu presented the same absorption bands in FTIR analysis, identifying the similarity between them.

Enzymatic hydrolysis increases ginsenoside content in Korean red ginseng (Panax ginseng CA Meyer) and its biotransformation under hydrostatic pressure.

BACKGROUND: Enzymatic hydrolysis and high hydrostatic pressure (HHP) are common processing techniques in the extraction of active compounds from food materials. The aim of this study was to investigate the effects of enzymatic hydrolysis combined with HHP treatments on ginsenoside metabolites in red ginseng. RESULTS: The yield and changes in the levels of polyphenol and ginsenoside were measured in red ginseng treated with commercial enzymes such as Ultraflo L, Viscozyme, Cytolase PCL5, Rapidase and Econase E at atmospheric pressure (0.1 MPa), 50 MPa, and 100 MPa. ß-Glucosidase activity of Cytolase was the highest at 4258.2 mg-1 , whereas Viscozyme showed the lowest activity at 10.6 mg-1 . Pressure of 100 MPa did not affect the stability or the activity of the ß-glucosidase. Treatment of red ginseng with Cytolase and Econase at 100 MPa significantly increased the dry weight and polyphenol content of red ginseng, compared with treatments at 0.1 MPa and 50 MPa (P < 0.05). The amounts of ginsenoside and ginsenoside metabolites derived from red ginseng processed using Cytolase were higher than those derived from red ginseng treated with the other enzymes. Treatment with Cytolase also significantly increased the skin and intestinal permeability of red ginseng-derived polyphenols. CONCLUSION: Cytolase could be useful as an enzymatic treatment to enhance the yield of bioactive compounds from ginseng under HHP. In addition, ginsenoside metabolites obtained by Cytolase hydrolysis combined with HHP are functional substances with increased intestinal and skin permeability. © 2019 Society of Chemical Industry.

Enzymatic preparation of fructooligosaccharides-rich burdock syrup with enhanced antioxidative properties

Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.

Cost-effective biodiesel production from wet microalgal biomass by a novel two-step enzymatic process.

In this study, a novel two-step enzymatic process was firstly established to produce microalgae biodiesel using wet Chlorella biomass. In the first hydrolysis step, to reduce energy consumption and effectively disrupt microalgal cell wall, among cellulase, hemicellulase, papain, lysozyme and pectinase, the highest hydrolysis efficiency (67.52%) was obtained by cellulase at pH 5.0 with enzyme dosage of 200 U/g dry biomass at 40 °C for 12 h. In the second transesterification step, compared with liquid CAL-A/B from Candida antarctica and PLA from Aspergillus oryzae, liquid lipase TL from Thermomyces lanuginosus achieved the highest biodiesel conversion at 81.15:1 (v/w) ethanol/g TFAs ratio in 78-83% water content with 100 PLU/g TFAs lipase loading at 25 °C for 48 h. Moreover, similar results were obtained with three Chlorella species by this process. Overall, this two-step enzymatic process was a green, low-energy and efficient method for cost-effective biodiesel production using wet microalgal biomass.

Lipase-mediated direct in situ ring-opening polymerization of ε-caprolactone formed by a chemo-enzymatic method.

A novel method to synthesize poly(ε-caprolactone) (PCL) through a three-step, lipase-mediated chemo-enzymatic reaction from cyclohexanone using an immobilized lipase from Trichosporon laibacchii (T. laibacchii) CBS5791 was developed. The immobilized preparation with 1280 U· g-1 used here was obtained by a method of purification and in situ immobilization where the crude intracellular lipase (cell homogenate) was subjected to partial purification by an aqueous two-phase system (ATPS) consisting of 12% (w/w) polyethylene glycol (PEG) 4000 and 13% (w/w) potassium phosphate (K2HPO4) and then in situ immobilization directly on diatomite from the top PEG-rich phase of ATPS. In this multi-step process, the ε-caprolactone (ε-CL) produced by lipase-mediated one-pot two-step chemo-enzymatic oxidation of cyclohexanone was directly subjected to in situ ring-opening polymerization (ROP) started by adding highly hydrophobic solvents. It is necessary to note that ε-CL synthesis and its subsequent ROP were catalyzed by the same lipase. The impact of various reaction parameters, e.g., solvent, cyclohexanone: hydrogen peroxide molar ratio, hydrogen peroxide forms and reaction temperature were investigated. Toluene was selected as a preferred solvent due to supporting the highest molecular weight (Mn = 2168) and moderate ε-CL conversion (65.42%). Through the optimization of reaction conditions, PCL was produced with a Mn of 2283 at 50 °C for 24 h. These results reveal that this lipase-mediated direct ring-opening polymerization of in situ formed ε-CL is an alternative route to the conventional synthesis of PCL.

Progress in One-pot Bioconversion of Cephalosporin C to 7-Aminocephalosporanic Acid.

BACKGROUND: Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-aminocephalosporanic acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste. METHODS: A two-step enzymatic process that utilized D-amino acid oxidase (DAAO) and 7-ß-(4carboxybutanamido)-cephalosporanic acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale. RESULTS: To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA. CONCLUSION: Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.

Mechanochemical synthesis of graphene oxide-supported transition metal catalysts for the oxidation of isoeugenol to vanillin.

Vanillin is one of the most commonly used natural products, which can also be produced from lignin-derived feedstocks. The chemical synthesis of vanillin is well-established in large-scale production from petrochemical-based starting materials. To overcome this problem, lignin-derived monomers (such as eugenol, isoeugenol, ferulic acid etc.) have been effectively used in the past few years. However, selective and efficient production of vanillin from these feedstocks still remains an issue to replace the existing process. In this work, new transition metal-based catalysts were proposed to investigate their efficiency in vanillin production. Reduced graphene oxide supported Fe and Co catalysts showed high conversion of isoeugenol under mild reaction conditions using H2O2 as oxidizing agent. Fe catalysts were more selective as compared to Co catalysts, providing a 63% vanillin selectivity at 61% conversion in 2 h. The mechanochemical process was demonstrated as an effective approach to prepare supported metal catalysts that exhibited high activity for the production of vanillin from isoeugenol.

Enzymatic process optimization for the in vitro production of isoprene from mevalonate.

BACKGROUND: As an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro. RESULTS: This study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0 µM of MVK, PMK, MVD; 10.0 µM of IDI, 80.0 µM of ISPS) could produce 6323.5 µmol/L/h (430 mg/L/h) isoprene in a 2 ml in vitro system. In a scale up process (50 ml) only using 1 balanced enzyme unit (0.5 µM of MVK, PMK, MVD; 1.0 µM of IDI, 8.0 µM of ISPS), the system could produce 302 mg/L isoprene in 40 h, which showed higher production rate and longer reaction phase with comparison of the in vivo control. CONCLUSIONS: By optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate.

Value addition of corn husks through enzymatic production of xylooligosaccharides

ABSTRACT Corn husks are the major wastes of corn industries with meagre economic significance. The present study was planned for value addition of corn husk through extraction of xylan, followed by its enzymatic hydrolysis into xylooligosaccharides, a pentose based prebiotic. Compositional analysis of corn husks revealed neutral detergent fibre 68.87%, acid detergent fibre 31.48%, hemicelluloses 37.39%, cellulose 29.07% and crude protein 2.68%. Irrespective of the extraction conditions, sodium hydroxide was found to be more effective in maximizing the yield of xylan from corn husks than potassium hydroxide (84% vs. 66%). Application of xylanase over the xylan of corn husks resulted into production of xylooligosaccharides with different degree of polymerization namely, xylobiose and xylotriose in addition to xylose monomer. On the basis of response surface model analysis, the maximum yield of xylobiose (1.9 mg/ml) was achieved with the enzymatic hydrolysis conditions of pH 5.8, temperature 44°C, enzyme dose 5.7U/ml and hydrolysis time of 17.5h. Therefore, the corn husks could be used as raw material for xylan extraction vis a vis its translation into prebiotic xylooligosaccharides.

Environmentally benign process for the preparation of antimicrobial α-methylene-ß-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass.

Tulipalin B (α-methylene-ß-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner.

Biotransformation of catechin and extraction of active polysaccharide from green tea leaves via simultaneous treatment with tannase and pectinase.

BACKGROUND: Green tea is a dietary source of bioactive compounds for human health. Enzymatic treatments induce the bioconversion of bioactive components, which can improve biological activities. In this study, we investigated the effect of simultaneous treatment with tannase and Rapidase on biotransformation of catechins and extraction of polysaccharide from green tea extract (GTE). RESULTS: Tannase and pectinase treatments induced the biotransformation of catechins and altered tea polysaccharide () content. The addition of GTE to the enzyme reaction resulted in a significant increase in degallated catechins, including gallic acid, a product of the tannase reaction (314.5-4076.0 µg mL(-1)) and a reduction in epigallocatechin gallate (EGCG). Biotransformation of catechins improved the radical scavenging activity of GTE. Pectinase treatment led to change of TPS composition in GTE by hydrolyzing polysaccharides. In addition, pectinase-driven hydrolysis in polysaccharides significantly increased TPS-induced Interleukin 6 (IL-6) production in macrophages. In particular, treatment of Rapidase (TPS-Ra) led to the highest IL-6 production among TPS samples, similar to treatment of highly purified pectinase (TPS-GTE), a positive control. CONCLUSION: Simultaneous processing with tannase and Rapidase can be an efficient method for the extraction of bioactive polysaccharides and biotransformation of catechins with enhanced radical scavenging activity from green tea.

Enzymatic process for the fractionation of baker's yeast cell wall (Saccharomyces cerevisiae).

ß-Glucans, homopolymers of glucose, are widespread in many microorganisms, mushrooms and plants. They have attracted attention because of their bioactive and medicinal functions. One important source of ß-glucans is the cell wall of yeasts, especially that of baker's yeast Saccharomyces cerevisiae. Several processes for the isolation of ß-glucans, using alkali, acid or a combination of both, result in degradation of the polymeric chains. In this paper, we have an enzymatic process for the isolation of glucans from yeast cell walls. As a result, ß-glucans were obtained in a yield of 18.0% of the original ratio in the yeast cell walls. Therefore, this isolation process gave a better yield and higher ß-glucan content than did traditional isolation methods. Furthermore, results showed that each extraction step of ß-glucan had a significant effects on its chemical properties.