Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins.

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.

Potential utility of bacterial protein nanoreactor for sustainable in-situ biocatalysis in wide range of bioprocess conditions.

Bacterial microcompartments (MCPs) are proteinaceous organelles that natively encapsulates the enzymes, substrates, and cofactors within a protein shell. They optimize the reaction rates by enriching the substrate in the vicinity of enzymes to increase the yields of the product and mitigate the outward diffusion of the toxic or volatile intermediates. The shell protein subunits of MCP shell are selectively permeable and have specialized pores for the selective inward diffusion of substrates and products release. Given their attributes, MCPs have been recently explored as potential candidates as subcellular nano-bioreactor for the enhanced production of industrially important molecules by exercising pathway encapsulation. In the current study, MCPs have been shown to sustain enzyme activity for extended periods, emphasizing their durability against a range of physical challenges such as temperature, pH and organic solvents. The significance of an intact shell in conferring maximum protection is highlighted by analyzing the differences in enzyme activities inside the intact and broken shell. Moreover, a minimal synthetic shell was designed with recruitment of a heterologous enzyme cargo to demonstrate the improved durability of the enzyme. The encapsulated enzyme was shown to be more stable than its free counterpart under the aforementioned conditions. Bacterial MCP-mediated encapsulation can serve as a potential strategy to shield the enzymes used under extreme conditions by maintaining the internal microenvironment and enhancing their cycle life, thereby opening new means for stabilizing, and reutilizing the enzymes in several bioprocess industries.

Process development of sugar beet enzymatic hydrolysis with enzyme recycling for soluble sugar production.

Enzymatic hydrolysis of sugar beets for achieving liquefaction and sugar release is a critical step for beet-ethanol production. An enzyme recycling process was developed in this study to reduce the economic uncertainty raised by the high costs of enzymes by reducing the fresh enzyme usage. A mixture of cellulases and pectinases was used in the beet hydrolysis. The hydrolysate was centrifuged and then processed through a 50 kDa molecular weight cut-off polyethersulfone membrane to recover enzymes from the liquid. Liquid enzyme recycling with 50% fresh enzyme addition achieved a similar liquefaction extent and sugar yield compared to the positive control with 100% fresh enzyme. Solid enzyme recycling showed a lower liquefaction efficiency, requiring at least 75% of fresh enzyme addition for a comparable liquefaction extent. Five sequential batches of hydrolysis with liquid enzyme recycling were successfully conducted to hydrolyze sugar beets with similar liquefaction extents and sugar yields.

High solid loading enzymatic hydrolysis of acetic acid-peroxide/acetic acid pretreated poplar and cellulase recycling.

High solid loading saccharification is the premise of preparing high-concentration sugar which is beneficial to bioethanol production, but the limited sugar concentration and high enzyme dosage are two challenges. In this work, the glucan-rich acetic acid-hydrogen peroxide/acetic acid (AC-HPAC)-pretreated poplar (85.8%) were prepared for enzymatic hydrolysis at 10%-40% solid loading and the strategies for reducing cellulase dosage were explored. Results showed that the maximum glucose concentration reached to 250.8 g/L at 40% solid loading, which was the highest concentration in previous literatures. As the solid loading was 20%, the addition of Tween 80 saved 50% of cellulase and the recycling of unhydrolyzed residue (0.2 g/g DM) saved another 25% of cellulase, resulting in 152.2 g/L of glucose concentration with yield of 79.9%. This work showed potential of poplar to produce the high concentration glucose solution with low enzyme loading through the recycling of enzyme bound onto unhydrolyzed residue.

Electrochemical biosensors based on multienzyme systems: Main groups, advantages and limitations - A review.

In the review, the principles and main purposes of using multienzyme systems in electrochemical biosensors are analyzed. Coupling several enzymes allows an extension of the spectrum of detectable substances, an increase in the biosensor sensitivity (in some cases, by several orders of magnitude), and an improvement of the biosensor selectivity, as showed on the examples of amperometric, potentiometric, and conductometric biosensors. The biosensors based on cascade, cyclic and competitive enzyme systems are described alongside principles of function, advantages, disadvantages and practical use for real sample analyses in various application areas (food production and quality control, clinical diagnostics, environmental monitoring). The complications and restrictions regarding the development of multienzyme biosensors are evaluated. The recommendations on the reasonability of elaboration of novel multienzyme biosensors are given.

Dialysis membrane enclosed laccase catalysis combines a controlled conversion rate and recyclability without enzyme immobilization.

Laccase is a versatile multicopper oxidase that holds great promise for many biotechnological applications. For such applications, it is essential to explore good biocatalytic systems for high activity and recyclability. The feasibility of membrane enclosed enzymatic catalysis (MEEC) for enzyme recycling with laccase was evaluated. The dialysis membrane enclosed laccase catalysis (DMELC) was tested for the conversion of the non-phenolic model substrate 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Trametes versicolor laccase was found to be completely retained by the dialysis membrane during the process. The ABTS total conversion after DMELC reached the same values as the batch reaction of the enzyme in solution. The efficiency of DMELC conversion of ABTS under different process conditions including shaking speed, temperature, ABTS concentration and pH was investigated. The repetitive dialysis minimally affected the activity and the protein content of the enclosed laccase. DMELC retained 70.3 ± 0.8% of its initial conversion after 5 cycles. The usefulness of MEEC extends to other enzymes with the benefit of superior activity of an enzyme in solution and the recyclability which is normally only obtained with immobilized enzymes.

Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach.

Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

Bases moleculares da absorção de nutrientes, tamponamento e fluxos de fluídos no intestino médio de Musca domestica / Molecular bases of nutrient absorption, buffering and fluid fluxes in the midgut of Musca domestica

O intestino dos insetos representa uma interface pouco protegida dos agentes externos. A identificação dos mecanismos moleculares envolvidos nos processos fisiológico-digestivos permite encontrar alvos potenciais para o controle de insetos. As moléculas envolvidas na absorção de nutrientes, tamponamento e geração de fluxos de água no intestino médio do inseto-modelo Musca domestica (Diptera Cyclorrhapha) foram identificadas. Para isso, foi feita uma análise da expressão gênica ao longo do intestino médio, a identificação e anotação de proteínas por bioinformática, a confirmação da localização apical das proteínas por análise proteômica de membranas microvilares purificadas e a determinação do papel de algumas das proteínas através de experimentos in vivo utilizando diferentes dietas, corantes e inibidores. A acidificação da região média é consequência da atividade anidrase carbônica que gera prótons que são bombeados por uma H+ V-ATPase apical acompanhados por cloreto transportado por um simporter K+Cl-. O K+ é recuperado por um canal de K+ e a homeostase dos cátions mantida pela Na+/K+-ATPase basolateral. O bicarbonato é eliminado basolateralmente em troca por cloreto por um antiporter. A acidificação é regulada diretamente por um antiporter Na+/H+ e indiretamente por uma proteína envolvida na homeostase do cobre. O muco protetor da região média é tamponado com bicarbonato gerado por uma anidrase carbônica com âncora de GPI e transportado por um antiporter Na+HCO3-/H+Cl-. O excesso de ácido é eliminado por um antiporter Na+/H+ situado na membrana basolateral. A alcalinização da região posterior ocorre pelo transporte apical de NH3 que sequestra os prótons luminais gerando amônio, junto à remoção de prótons em simporte com aminoácidos e peptídeos. A acidificação intracelular, consequência da entrada de aminoácidos com prótons, é regulada por uma H+ V-ATPase basolateral. A geração de fluxos de água é consequência da atividade conjunta de simporters NKCC e KCC ajudados pelas aquaporinas. A inibição dos simporters com inibidores específicos mostrou que o contrafluxo de água está envolvido na reciclagem da enzima tripsina. Por último, o principal lugar de absorção nutrientes no intestino médio é a região posterior, a exceção do cobre que é absorvido na região média

The gut of insects is a less protected interphase against external agents. The identification of the molecular mechanisms involved in physiological-digestive processes allows one to find potential targets for insect control. The molecules involved in nutrient absorption, buffering and fluid fluxes in the midgut of the insect-model M. domestica (Diptera Cyclorrhapha) were identified. For this, gene expression along the midgut was analyzed; proteins were identified and annotated by bioinformatics; apical localization of proteins was confirmed by proteomics of purified microvillar membranes; the role of proteins was confirmed by in vivo experiments using different diets, dyes and inhibitors. Middle midgut acidification occurs by the action of an apical H+ V-ATPase with protons coming from carbonic anhydrase activity accompanied by chloride transported with potassium by a K+Cl- symporter. Potassium is recovered by a potassium channel, and cation homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is directly regulated by a Na+/H+ antiporter and indirectly by a copper homeostasis protein. Mucus protecting epithelium is neutralized with bicarbonate generated by a GPI-ancored carbonic anhydrase and transported by a Na+HCO3-/H+Cl- antiporter. Intracellular acidification is avoided by a basolateral Na+/H+ antiporter. Posterior midgut alkalization occurs by the action of an apical ammonia transporter and proton amino acid (and peptide) symporters. Intracellular acid is eliminated by a basolateral H+ V-ATPase. Fluid fluxes are generated by K+Cl- and Na+Cl-Cl- symporters helped by aquaporins. Inhibition of these symporters showed that the countercurrent flux of water allows trypsin recycling. Finally, posterior midgut is the main location of nutrient absorption, except for copper which is absorbed in the middle midgut

Developing fast enzyme recycling strategy through elucidating enzyme adsorption kinetics on alkali and acid pretreated corn stover.

BACKGROUND: Although various pre-treatment methods have been developed to disrupt the structure of lignocellulosic biomass, high dosage of cellulases is still required to hydrolyze lignocellulose to fermentable sugars. Enzyme recycling via recycling unhydrolyzed solids after enzymatic hydrolysis is a promising strategy to reduce enzyme loading for production of cellulosic ethanol. RESULTS: To develop effective enzyme recycling method via recycling unhydrolyzed solids, this work investigated both enzymatic hydrolysis kinetics and enzyme adsorption kinetics on dilute acid and dilute alkali pre-treated corn stover (CS). It was found that most of the hydrolysable biomass was hydrolyzed in the first 24 h and about 40% and 55% of the enzymes were adsorbed on unhydrolyzed solids for dilute alkali-CS and dilute acid-CS, respectively, at 24 h of enzymatic hydrolysis. Lignin played a significant role in such adsorption and lignin materials derived from dilute acid-CS and dilute alkali-CS possessed different enzyme adsorption properties. Enzyme recycling was performed by recycling unhydrolyzed solids after 24 h enzymatic hydrolysis for five successive rounds, and successfully reduced 40% and 50% of the enzyme loadings for hydrolysis of dilute alkali-CS and for hydrolysis of dilute acid-CS, respectively. CONCLUSIONS: This study presents that the enzymes adsorbed on the unhydrolyzed solids after short-time hydrolysis could be recycled effectively for efficient enzymatic hydrolysis. Lignin derived from dilute acid-CS has higher enzyme adsorption capacity than the lignin derived from dilute alkali-CS, which led to more enzymes recycled. By applying the enzyme recycling strategy developed in this study, the enzyme dosage needed for effective cellulose hydrolysis can be significantly reduced.

Electrochemical aptasensor for multi-antibiotics detection based on endonuclease and exonuclease assisted dual recycling amplification strategy.

An ultrasensitive electrochemical aptasensor for multiplex antibiotics detection based on endonuclease and exonuclease assisted dual recycling amplification strategy was proposed. Kanamycin and chloramphenicol were selected as candidates. Firstly, aptamers of the antibiotics were immobilized on bar A and then binding with their endonuclease labeled complementary DNA strands to construct enzyme-cleavage probes. Secondly, The nano zirconium-metal organic framework (NMOF) particles with 1,4-benzene-dicarboxylate (BDC) as linker was defined as UiO-66. And its updated version, hierarchically porous UiO-66 (HP-UIO-66) decorated with different electroactive materials as signal tags were synthesized. Then they were immobilized on bar B linked by two duplex DNA strands which can be specifically cleaved by corresponding enzyme-cleavage probes in bar A. Once targets were introduced into system, aptamers can capture them and then release enzyme-cleavage probes. In the presence of exonuclease-I, exonuclease assisted target recycling amplification was triggered and more enzyme-cleavage probes were released into solution. The probes can trigger endonuclease assisted recycles and repeatedly cleave their corresponding duplex DNA strands on bar B then released numerous signal tags into supernatant. Thus two recycling amplification was performed in the system. Finally, MB and Fc in the signal tags were detected by square wave voltammetry after removing bar A/B and the current intensities were correspondent with the concentration of KANA and CAP respectively. Under the optimum condition, the limits of detection for the KANA and CAP were 35fM and 21fM respectively with a wide linear range from 1 × 10-4 to 50nM. This dual recycling amplification detection system exhibited high sensitivities and specificity. It can be easily extended to detect other targets if changing the corresponding aptamers and has potential application values for screening of multiplex antibiotics residues in food safety.

Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc.

Capture and Recycling of Sortase†A through Site-Specific Labeling with Lithocholic Acid.

Enzyme-mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small-molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N-terminally labeled with lithocholic acid (LA)-an inexpensive bile acid that exhibits strong binding to ß-cyclodextrin (ßCD). Capture and recycling of the LA-Pro-SrtA 7M conjugate was achieved using ßCD-modified sepharose resin. The LA-Pro-SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.

The use of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk.

The aim of this research was to determine the potential of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk. The lipase was immobilised on hydrophobic resin (Toyopearl® Butyl) and used to hydrolyse milk lipids in a batch reactor. The lipase was stable when immobilised and there was no significant resin fouling or enzyme inhibition between cycles. Eight cycles were achieved before the hydrolysis rate dropped significantly because of physical losses of the immobilised lipase. The immobilised lipase showed the highest specificity towards short-chain fatty acids butanoic and hexanoic acids, the main dairy product flavour and odour compounds. Based on the performance of the reactor, and the ability of the lipase to alter free fatty acid composition and sensory characteristics of milk, the immobilised salmon lipase has potential applications in developing dairy products with unique flavours.

Celluclast and Cellic® CTec2: Saccharification/fermentation of wheat straw, solid-liquid partition and potential of enzyme recycling by alkaline washing.

The hydrolysis/fermentation of wheat straw and the adsorption/desorption/deactivation of cellulases were studied using Cellic(®) CTec2 (Cellic) and Celluclast mixed with Novozyme 188. The distribution of enzymes - cellobiohydrolase I (Cel7A), endoglucanase I (Cel7B) and ß-glucosidase - of the two formulations between the residual substrate and supernatant during the course of enzymatic hydrolysis and fermentation was investigated. The potential of recyclability using alkaline wash was also studied. The efficiency of hydrolysis with an enzyme load of 10 FPU/g cellulose reached >98% using Cellic(®) CTec2, while for Celluclast a conversion of 52% and 81%, was observed without and with ß-glucosidase supplementation, respectively. The decrease of Cellic(®) CTec2 activity observed along the process was related to deactivation of Cel7A rather than of Cel7B and ß-glucosidase. The adsorption/desorption profiles during hydrolysis/fermentation revealed that a large fraction of active enzymes remained adsorbed to the solid residue throughout the process. Surprisingly, this was the case of Cel7A and ß-glucosidase from Cellic, which remained adsorbed to the solid fraction along the entire process. Alkaline washing was used to recover the enzymes from the solid residue. This method allowed efficient recovery of Celluclast enzymes; however, this may be achieved only when minor amounts of cellulose remain present. Regarding the Cellic formulation, neither the presence of cellulose nor lignin restricted an efficient desorption of the enzymes at alkaline pH. This work shows that the recycling strategy must be customized for each particular formulation, since the enzymes found e.g. in Cellic and Celluclast bear quite different behaviour regarding the solid-liquid distribution, stability and cellulose and lignin affinity.

Reducing ß-glucosidase supplementation during cellulase recovery using engineered strain for successive lignocellulose bioconversion.

Enzyme recycling by re-adsorption is one of the primary methods for reducing enzyme usage in lignocellulose conversion. This work proposes the combination of an engineered yeast strain that expresses ß-glucosidase with enzyme recycling to reduce the amount of supplemented ß-glucosidase in enzyme recycling experiments. Using the engineered strain, a slight increase in ethanol concentration was obtained after a 96-h fermentation of pretreated corncobs. Ethanol concentrations increased by 34.7% and 62.7% in the following two recycle rounds using the engineered strain compared with those using its parental strain without ß-glucosidase addition. Furthermore, with the addition of ß-glucosidase at 30CBU/g cellulose, the ethanol concentration after two recycle rounds exceeded 90% of that observed in the first SSF round with the engineered strain at a high initial cellulase loading of 45FPU/g cellulose.

Increased enzymatic hydrolysis of sugarcane bagasse from enzyme recycling.

BACKGROUND: Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings. RESULTS: It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes. CONCLUSIONS: Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.

Recycling cellulases for cellulosic ethanol production at industrial relevant conditions: potential and temperature dependency at high solid processes.

Different versions of two commercial cellulases were tested for their recyclability of enzymatic activity at high dry matter processes (12% or 25% DM). Recyclability was assessed by measuring remaining enzyme activity in fermentation broth and the ability of enzymes to hydrolyse fresh, pretreated wheat straw. Industrial conditions were used to study the impact of hydrolysis temperature (40 or 50°C) and residence time on recyclability. Enzyme recycling at 12% DM indicated that hydrolysis at 50°C, though ideal for ethanol yield, should be kept short or carried out at lower temperature to preserve enzymatic activity. Best results for enzyme recycling at 25% DM was 59% and 41% of original enzyme load for a Celluclast:Novozyme188 mixture and a modern cellulase preparation, respectively. However, issues with stability of enzymes and their strong adsorption to residual solids still pose a challenge for applicable methods in enzyme recycling.

Enzymatic hydrolysis, adsorption, and recycling during hydrolysis of bagasse sulfite pulp.

The high costs of enzymatic hydrolysis along with the high enzyme dosage are often considered as the major bottlenecks in lignocellulosic bioconversion. This study investigated the hydrolysis efficiency, cellulase adsorption and enzyme recycling during the hydrolysis of bagasse sulfite pulp (BSP). After 48 h of hydrolysis, more than 70% of the cellulose was hydrolyzed, while the protein concentration and cellulase activity in solution remained 31% and 17% of the initial value, respectively. The cellulase adsorption on the fresh BSP was better fitted by a Sips model, suggesting the occurrence of a multilayer adsorption at low cellulase concentration and monolayer adsorption at high concentration on the BSP surfaces. Desorption profile studies showed that the optimum desorption condition was at pH 4.8 and 40 °C. Moreover, considering the limited ability to desorption, directly empolying the bound enzyme with residual substrate is more effective method to recover cellulase during the hydrolysis of BSP.