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Metal–organic frameworks (MOFs), with large specific surface area and tunable porosity, have gained lots of attention for immobilizing enzymes. However, the intrinsic open channels of most reported MOFs are generally smaller than 2 nm, which significantly prevents the passage of enzymes, and the diffusion efficiency of substrates and products. Here we report a new hierarchical micro-mesoporous zeolitic imidazolate framework-8 (ZIF-8) with core-shell superstructure (HZIF-8) using colloidal hydrated zinc sulfate (ZnSO4•7H2O) as a soft template for enzyme immobilization. The ZnSO4•7H2O forms an aggregation of colloids due to the self-conglobation effect in methanol, which affords a soft template for the formation of HZIF-8. Cytochrome C (Cyt C) was immobilized in interior of HZIF-8 through entrapment during the formation of HZIF-8. The resultant immobilized Cyt C (Cyt C@HZIF-8) exhibited 4-fold and 3-fold higher activity than free Cyt C and Cyt C encapsulated in conventional microporous ZIF-8 (Cyt C@ZIF-8), respectively. Meanwhile, the Km value of Cyt C@HZIF-8 significantly decreased due to the presence of mesopores compared with Cyt C@ZIF-8, indicating enhanced substrate affinity. After 7 cycles, Cyt C@HZIF-8 still maintained 70% of its initial activity whereas Cyt C@ZIF-8 only retained 10% of its initial activity. Moreover, the obtained HZIF-8 showed outstanding performance in co-immobilization of multi-enzyme for the detection of glucose.

In this study, the surfactant activated lipase from Aspergillus oryzae was used to prepare magnetic hybrid nanoflowers by embedding Fe3O4 magnetic nanoparticles (MNPs) into hybrid nanoflowers (MhNF). Meanwhile, MNPs were integrated into the activated lipase hybrid nanoflowers through covalent cross-linking (cross-linked-MhNF). Activity recovery of the MhNF and cross-linked-MhNF was 190% and 174%, respectively. However, activity recovery of lipase hybrid nanoflowers (hNF) only retained 77%. Furthermore, the MhNF displayed higher Kcat/Km value than free lipase, indicating high catalytic efficiency of MhNF. Compared with free lipase, the MhNF exhibited high tolerance against methanol and storage stability. Furthermore, the MhNF could be easily recycled by magnet without obvious activity loss. After reusing for 10 cycles, the activated hNF only retained 26% activity of its original activity, while the MhNF still maintained 84% activity of its initial activity, indicating excellent reusability. In the reaction of producing biodiesel from soybean oil, the MhNF-catalyzed biodiesel yield reached 88%, while the free enzyme was only 69%. The yield of biodiesel catalyzed by MhNF was maintained at 76% even after 6 consecutive cycles.

Nanostructured materials constitute an interesting and novel class of support matrices for the immobilization of peroxidase enzymes. Owing to the high surface area, robust mechanical stability, outstanding optical, thermal, and electrical properties, nanomaterials have been rightly perceived as immobilization matrices for enzyme immobilization with applications in diverse areas such as nano-biocatalysis, biosensing, drug delivery, antimicrobial activities, solar cells, and environmental protection. Many nano-scale materials have been employed as support matrices for the immobilization of different classes of enzymes. Nanobiocatalysts, enzymes immobilized on nano-size materials, are more stable, catalytically robust, and could be reused and recycled in multiple reaction cycles. In this review, we illustrate the unique structural/functional features and potentialities of nanomaterials-immobilized peroxidase enzymes in different biotechnological applications. After a comprehensive introduction to the immobilized enzymes and nanocarriers, the first section reviewed carbonaceous nanomaterials (carbon nanotube, graphene, and its derivatives) as a host matrix to constitute robust peroxidases-based nanobiocatalytic systems. The second half covers metallic nanomaterials (metals, and metal oxides) and some other novel materials as host carriers for peroxidases immobilization. The next section vetted the potential biotechnological applications of the resulted nanomaterials-immobilized robust peroxidases-based nanobiocatalytic systems. Concluding remarks, trends, and future recommendations for nanomaterial immobilized enzymes are also given.

Multienzymatic conversion of carbon dioxide (CO2) into chemicals has been extensively studied. However, regeneration and reuse of co-factor are still the main problems for the efficient conversion of CO2. In this study, a nanoscale multienzyme reactor was constructed by encapsulating simultaneously carbonic anhydrase (CA), formate dehydrogenase (FateDH), co-factor (NADH), and glutamate dehydrogenases (GDH) into ZIF-8. In the multienzyme reactors, cationic polyelectrolyte (polyethyleneimine, PEI) was doped in the ZIF-8 by dissolving it in the precursors of ZIF-8. Co-factor (NADH) was anchored in ZIF-8 by ion exchange between PEI (positive charge) and co-factor (negative charge), and regenerated through GDH embedded in the ZIF-8, thus keeping high activity of FateDH. Activity recovery of FateDH in the multienzyme reactors reached 50%. Furthermore, the dissolution of CO2 in the reaction solution was increased significantly by the combination of CA and ZIF-8. As a result, the nanoscale multienzyme reactor exhibited superior capacity for conversion of CO2 to formate. Compared with free multienzyme system, formate yield was increased 4.6-fold by using the nanoscale multienzyme reactor. Furthermore, the nanoscale multienzyme reactor still retained 50% of its original productivity after 8 cycles, indicating excellent reusability.

Polyvinyl alcohol (PVA) is a synthetic polymer that is difficult to degrade in nature. In this study, we synthesized PVA-degrading enzymes (PVAase)-Cu3(PO4)2 hybrid nanoflowers by using crude PVAase from Bacillus niacini for PVA degradation. The influences of PVAase concentration, Cu2+ concentration, and incubation time on the nucleation and activity of the PVAase hybrid nanoflower were investigated. The maximal activity recovery of the PVAase hybrid nanoflower was approximately 85% at 0.25 mg/mL of PVAase, 0.36mM Cu2+, and 72 h incubation time. The optimum temperature and pH of PVAase did not change before and after immobilization. Compared with free PVAase, the PVAase hybrid nanoflower showed high thermal stability and storage stability. Additionally, the PVAase hybrid nanoflower displayed excellent reusability after eight cycles and promising PVA degradability, indicating its potential application in PVA degradation.

e-Poly-L-lysine (e-PL) is a natural antimicrobial cationic peptide. Antimicrobial activity of e-PL is closely related to its molecular weight (Mw). However, the antimicrobial mechanisms of e-PL with different Mws are still vague. In this study, Saccharomyces cerevisiae was used as the model system to analyze the mechanism from these three aspects: cell wall, cell membrane, and metabolism. The results showed that high-Mw e-PL (1-3 kDa and >3 kDa) and commercial e-PL product caused cell wall lesions, and significantly improved cell membrane permeability compared to low-Mw e-PL ( 3 kDa) and product displayed higher inhibition effect on the glycolysis pathway and tricarboxylic cycle than that of low-Mw e-PL (

Biological metal-organic frameworks (bioMOFs) are a new subclass of the MOF family. In comparison with traditional MOFs, the bioMOFs are made of multifunctional biologically related ligands (bio-ligand) and metal ions. The bio-ligands confer biological compatibility for traditional MOFs, thus providing many opportunities for a wide array of biological applications. This review highlights the recent advances in the synthesis of bioMOFs comprising multifunctional bio-ligands and metal ions. These bio-ligands include nucleobases, amino acids, peptides, proteins, cyclodextrin, saccharides, and other biomolecules. Furthermore, the potential bio-applications of bioMOFs in several fields such as biomedicine, biosensing and bioimaging, antimicrobial applications, biomimetic catalysis, chiral separation, and environmental protection are also demonstrated.

Zeolitic imidazole frameworks (ZIFs) with tunable pore sizes and high surface areas have recently used as an effective support for immobilizing enzymes. However, the instability in the aqueous acidic environment has limited their practical applications in some cases. In this work, we develop a novel catalase/ZIFs composite with mesoporous silica shell (mSiO2@CAT/ZIFs) via co-precipitation, and controlled self-assembly of silanes. During preparation, the cetyltrimethylammonium bromide induced the formation of the mesostructured silica layer on the outer surface of CAT/ZIFs. The resultant mSiO2@CAT/ZIFs exhibited high activity recovery (92%). Compared with the conventional CAT/ZIFs and free CAT, mSiO2@CAT/ZIFs exhibited excellent acid resistance. For example, after 30 min in acetate buffer solution (pH 3.0), the CAT/ZIFs and free CAT almost lost activity whereas the mSiO2@CAT/ZIFs still retained 35% of original activity. Meanwhile, the thermostability of the mSiO2@CAT/ZIFs was enhanced significantly compared with conventional CAT/ZIFs. In addition, the mSiO2@CAT/ZIFs displayed excellent storage stability, and retained 60% of its initial activity after 15 days storage period. Furthermore, the mSiO2@CAT/ZIFs could maintain 70% of its initial activity after 8 continuous uses, demonstrating superior reusability than the free CAT and CAT/ZIFs. These results demonstrated that the mSiO2@CAT/ZIFs are potential for practical applications even in the acidic environment.

Bacterial cellulose (BC) has numerous excellent properties but the absence of antibacterial activity restricts its applications in biomedical field. Therefore, in order to introduce the antibacterial characteristics into BC; herein, a facile method for incorporation of ZnO nanoparticles (ZnO-NPs) is presented. BC films were first immersed in zinc nitrate solution, followed by treating with NaOH solution, the BC loaded ZnO nanocomposite films were dried by a sheet former instrument at 80 °C for 20 min. The obtained BC/ZnO nanocomposites were characterized by different techniques. XRD results showed the hexagonal wurtzite structure of ZnO-NPs while FE-SEM results displayed the particle size of ZnO-NPs was ranging from 70 to 100 nm. Thermogravimetric study revealed the thermal stability of nanocomposite films. The nanocomposite exhibited photocatalytic activity and revealed 91% degradation of methyl orange (MO) under UV-irradiation within 2 h. Moreover, the nanocomposites demonstrated significant UV-blocking properties and showed antibacterial activities against tested Gram-positive and Gram-negative bacterial strains. This work provides a simple and novel method for the synthesis of BC/ZnO nanocomposite as a functional biomaterial.

Background As a natural antibacterial cationic peptide, ε-poly-l-lysine (ε-PL) is applied as a food preservative. However, the mechanism of ε-PL against Staphylococcus aureus (S. aureus) has not been elucidated. Especially, its antimicrobial mechanism at the metabolomics has not been yet thoroughly described. Results This work aimed at clarifying the antibacterial activity and mechanism of ε-PL against S. aureus. Effects of ε-PL with different concentration on cell morphology, cell wall, and membrane integrity were investigated. Furthermore, the effect of ε-PL on metabolite properties of S. aureus was also studied. The results revealed that ε-PL disrupted the cell wall and membrane integrity of treated cells. ε-PL induced the structural change of peptidoglycan in cell wall, causing cell wall more fragile. Meanwhile, the permeability of the S. aureus cell membrane was increased by ε-PL. More importantly, ε-PL with different concentration could cause different effects on metabolic pathways of S. aureus. ε-PL with high concentration could directly restrain the central carbon metabolism. However, ε-PL with low concentration could only inhibit the glycolytic pathway. Conclusion These results showed that the antimicrobial mechanism of ε-PL against S. aureus was a synergistic action.

In this study, carbonic anhydrase (CA, EC 4.2.1.1) molecules were embedded into metal-organic frameworks (MOFs) via co-precipitation (CA@ZIF-8), and then these CA@ZIF-8 nanocomposites were encapsulated in the poly(vinyl alcohol) (PVA)-chitosan (CS) hydrogel networks to prepare CA@ZIF-8-PVA-CS composite hydrogels (PVA/CS/CA@ZIF-8) with high activity, stability, and reusability. The immobilization efficiency of CA was greater than 70%, suggesting the high immobilization efficiency. The prepared PVA/CS/CA@ZIF-8 composite membranes displayed excellent higher stability against a high temperature, denaturants, and acid than free CA and CA@ZIF-8. Furthermore, these membranes exhibited an excellent performance for CO2 capture. The amount of calcium carbonate obtained by PVA/CS/CA@ZIF-8 hydrogel membranes was 20- and 1.63-fold than free CA and CA@ZIF-8 composites, respectively. Furthermore, the hydrogel membranes exhibited superior reusability and mechanical strength. The hydrogel membrane maitained 50% of its original activity after 11 cycles. However, CA@ZIF-8 completely lost activity. These results indicated that the PVA/CS/CA@ZIF-8 membranes can be efficiently applied to capture CO2 sequestration.

ε-Polylysine (ε-PL), a natural preservative with broad-spectrum antimicrobial activity, has been widely used as a green food additive, and it is now mainly produced by Streptomyces in industry. In the previous study, strain 6#-7 of high-yield ε-PL was obtained from the original strain TUST by mutagenesis. However, the biosynthesis mechanism of ε-PL in 6#-7 is still unclear. In this study, the metabolomic analyses of the biosynthesis mechanism of ε-PL in both strains are investigated. Results show that the difference in metabolisms between TUST and 6#-7 is significant. Based on the results of both metabolomic and enzymatic activities, a metabolic regulation mechanism of the high-yield strain is revealed. The transport and absorption capacity for glucose of 6#-7 is improved. The enzymatic activity benefits ε-PL synthesis, such as pyruvate kinase and aspartokinase, is strengthened. On the contrary, the activity of homoserine dehydrogenase in the branched-chain pathways is decreased. Meanwhile, the increase of trehalose, glutamic acid, etc. makes 6#-7 more resistant to ε-PL. Thus, the ability of the mutagenized strain 6#-7 to synthesize ε-PL is enhanced, and it can produce more ε-PLs compared with the original strain. For the first time, the metabolomic analysis of the biosynthesis mechanism of ε-PL in the high-yield strain 6#-7 is investigated, and a possible mechanism is then revealed. These findings provide a theoretical basis for further improving the production of ε-PL.

Metal-organic frameworks (MOFs) have been emerged as a promising support for immobilizing enzymes owing to the tunable porosity, high surface area, and structural diversity. However, most of these possess nanometer size and small pores, which are difficult to recover them from the reaction medium and present low immobilization efficiency and protein loading capacity, and high substrate diffusion limitations. Herein, a novel magnetic amino-functionalized zeolitic imidazolate framework-8 (ZIF-8) with 3D highly ordered macroporous structure was synthesized using the assembled polystyrene (PS) nanosphere monoliths as a template. Subsequently, catalase (CAT) molecules were immobilized on the surface of macroporous magnetic ZIF-8 and inside the macropores by precipitation, covalent binding and cross-linking. The resultant immobilized CAT showed high immobilization efficiency (58%) and protein loading capacity (29%), leading to 500% higher activity than the immobilized CAT on ZIF-8 (CAT/ZIF-8). Meanwhile, the immobilized CAT could be easily recovered with a magnet without obvious activity loss. The traditional CAT/ZIF-8 lost its activity after 6 cycles, whereas, the immobilized CAT retained 90% activity of its initial activity after reusing for 8 cycles, indicating excellent reusability. In conclusion, this study provides a facile and efficient approach to immobilize enzymes on/in MOFs with enhanced activity and excellent recyclability.

The Phenylketonuria (PKU) is an inborn defect of phenylalanine (Phe) metabolism, in which Phe accumulated in the blood causing alterations at the central nervous system. Therefore, the detection of PKU is very important for the early diagnosis of PKU patients. However, existing tests for PKU are time-consuming and require high-resource laboratories. In this study, a novel paper-based biosensor based on phenylalnine ammonia lyase (PAL) hybrid nanoflowers was constructed that provides a semi-quantitative output of the concentration of Phe from urine samples. PAL@Ca3(PO4)2 hybrid nanoflowers (PAL@NF) were first prepared using PAL and Ca2+. Synthesis conditions of the PAL@NF on the formation of the PAL@NF were optimized. The PAL@NF exhibited 90% activity recovery under optimal condition. Compared with free PAL, the PAL@NF displayed good storage stability and increased tolerance to proteolysis. After five consecutive operating cycles, the PAL@NF still retained 73% of its initial activity, indicating excellent reusability. Furthermore, the paper-based biosensor was able to detect Phe concentration in urine samples, and exhibited good linearity to the Phe concentrations in the range from 60 to 2400 μM and the response time was only about 10 min. Therefore, the paper-based biosensor can be a promising candidate as a biosensor for the detection of PKU.

Pectinases are the emerging enzymes of the biotechnology industry with a 25% share in the worldwide food and beverage enzyme market. These are green and eco-friendly tools of nature and hold a prominent place among the commercially produced enzymes. Pectinases exhibit applications in various industrial bioprocesses, such as clarification of fruit juices and wine, degumming, and retting of plant fibers, extraction of antioxidants and oil, fermentation of tea/coffee, wastewater remediation, modification of pectin-laden agro-industrial waste materials for high-value products biosynthesis, manufacture of cellulose fibres, scouring, bleaching, and size reduction of fabric, cellulosic biomass pretreatment for bioethanol production, etc. Nevertheless, like other enzymes, pectinases also face the challenges of low operational stability, recoverability, and recyclability. To address the above-mentioned problems, enzyme immobilization has become an eminently promising approach to improve their thermal stability and catalytic characteristics. Immobilization facilitates easy recovery and recycling of the biocatalysts multiple times, leading to enhanced performance and commercial feasibility.In this review, we illustrate recent developments on the immobilization of pectinolytic enzymes using polymers and nanostructured materials-based carrier supports to constitute novel biocatalytic systems for industrial exploitability. The first section reviewed the immobilization of pectinases on polymers-based supports (ca-alginate, chitosan, agar-agar, hybrid polymers) as a host matrix to construct robust pectinases-based biocatalytic systems. The second half covers nanostructured supports (nano-silica, magnetic nanostructures, hybrid nanoflowers, dual-responsive polymeric nanocarriers, montmorillonite clay), and cross-linked enzyme aggregates for enzyme immobilization. The biotechnological applications of the resulted immobilized robust pectinases-based biocatalytic systems are also meticulously vetted. Finally, the concluding remarks and future recommendations are also given.

Lipase-inorganic hybrid nanoflowers were prepared using Ca3(PO4)2 as the inorganic component and lipase from Aspergillus oryzae (A. oryzae) as the organic component. The influences of metal ions with different valence, various additives (surfactant), and synthesis conditions on the activity of the lipase hybrid nanoflowers were systematically investigated. Results revealed that the valence state of metal ions played an important role on the shape and activity of lipase hybrid nanoflowers. The synthesized lipase hybrid nanoflowers using bivalence metal ions (Ca2+, Mn2+, and Zn2+) as the inorganic components exhibited relative high activity. However, very low activities were observed in the lipase hybrid nanoflowers using univalent metal ions (Ag+) or trivalent metal ions (Al3+, Fe3+). More importantly, Ca2+ not only induced self-assemble of lipase hybrid nanoflowers, but also activated the enzyme activity by inducing conformational changes in lipase from A. oryzae. As a result, lipase/Ca3(PO4)2 hybrid nanoflowers (hNF-lipase) exhibited the high activity. The hNF-lipase displayed 9, 12, and 61 folds higher activity than lipase/Ag3PO4 hybrid nanoflowers, lipase/AlPO4 hybrid nanoflowers, and lipase/FePO4 nanoflowers, respectively. Compared with free lipase, the hNF-lipase displayed 172 % increase in activities by using 0.15 mM Tween-80 as an activity inducer (activated hNF-lipase). Furthermore, the hNF-lipase and activated hNF-lipase exhibited increased stability against high temperature and denaturant, and had good storage stability and reusability.

CO2 capture by immobilized carbonic anhydrase (CA) has become an alternative and environmental friendly approach in CO2 sequestration technology. However, the immobilized CA usually exhibits low CO2 sequestration efficiency due to no gas adsorption function for the conventional CA supports. Metal organic frameworks (MOFs) are an excellent material for gas adsorption and enzyme immobilization. Herein, a combined immobilization system of CA and ZIF-8 with cruciate flower-like morphology for CO2 adsorption was prepared for the first time by adsorbing CA onto ZIF-8. The immobilization efficiency was greater than 95%, and the maximum activity recovery reached 75%, indicating the highly efficient immobilization process. The resultant CA@ZIF-8 composites exhibited outstanding thermostability, the tolerance against denaturants, and reusability compared with free CA. Furthermore, we demonstrated for the first time that the shape of ZIF-8 could be controlled by adjusting concentrations of Zn2+ ions at the high concentration of 2-methylimidazole (1 M). More importantly, we also demonstrated the applicability of the CA@ZIF-8 composites to the sequestration of CO2 in carbonate minerals. The yields of the CaCO3 obtained by using CA@ZIF-8 composites were 22-folds compared to free CA. Thus, this CA@ZIF-8 composite can be successfully used as a robust biocatalyst for sequestration of CO2.

Enzyme immobilization on the external surface of solid supports is a commonly adopted method to improve stability and reuse for continuous operations, which, however, is prone to cause the enzyme denaturation due to no carriers protection. Herein, we describe enzyme-shielding strategy to prepare hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of immobilized enzymes on Fe3O4/silica core-shell nanospheres to grow a protective silica layer. The silica shell around the immobilized enzyme particles provides a “shield” to protect from biological, thermal and chemical degradation for enzyme. As a result, the recycling of the immobilized catalase with a protective silica layer was improved remarkably compared with immobilized catalase without a protective silica layer. The immobilized catalase with a protective silica layer still remained 70% of their original activity after 9 cycles, whereas the immobilized catalase without a protective silica layer only retained 20% of their original activity. Moreover, the immobilized catalase with a protective silica layer exhibited significantly enhanced resistance to denaturing stresses (such as proteolytic agent, denaturants, and heat). Therefore, the enzyme-shielding strategy showed promising applications for preparing obtain stable and recycled nanobiocatalyst.

Non-small cell lung cancer (NSCLC) is one of the most prevailing malignancies worldwide. It has been previously shown that wogonoside exerts anti-tumor activities in various kinds of human cancers. But its role in NSCLC remains elusive. In the present study, we determined the anti-tumor effect of wogonoside in human NSCLC A549 cells. We found that wogonoside effectively inhibits A549 cell viability through inducing cell cycle arrest and apoptosis. Moreover, administration of wogonoside by intraperitoneal injection inhibits the growth of A549 cell xenografts in athymic nude mice. Additionally, mitochondrial membrane potential was disrupted and cytochrome c was released to cytosol in the wogonoside-treated A549 cells. Finally, we found that AMPK/mTOR signaling might be implicated in the anti-NSCLC efficacy of wogonoside. Therefore, we may assume that wogonoside may be considered as a potential therapeutic agent for the treatment of NSCLC.

ε-Poly-l-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the antimicrobial mechanism of ε-PL against Saccharomyces cerevisiae (S. cerevisiae) is only vaguely described. Especially, it is widely accepted that membrane disruption is its main antimicrobial mode of action, but its effect on the cell wall remains unclear. In this study, the effects of ε-PL on cell wall of S. cerevisiae were investigated, and the possible action mode of ε-PL on the cell wall was discussed. The results showed that ε-PL affected significantly the cell wall composition such as β-1, 3-glucan, mannosylphosphate and chitin, and caused cell wall more fragile. The cell wall permeability was significantly increased. Furthermore, ε-PL induced the intracellular accumulation of reactive oxygen species (ROS), as well as lead to DNA fragmentation. These results indicate that ε-PL may have a complicated antimicrobial mode of action with multi-target mechanisms against S. cerevisiae cells.

Metal-organic frameworks (MOFs) are a type of porous material that have tunable porosity, desirable functionality, extremely high surface area, and chemical/thermal stability. MOFs consist of metal containing nodes and organic ligands linked through coordination bonds. Owing to the unique properties of MOFs, there is considerable interest in using them as a potential matrix for enzyme immobilization. Recent studies have focused on developing enzyme-MOF composites with potential applications. Many MOF-enzyme composites exhibit excellent catalytic performance, far outperforming free enzymes in many aspects. This review summarizes recent developments in enzyme-MOF composites with special emphasis on novel synthesizing strategies, process optimization, and improvement of catalytic performance of the enzyme-MOF composites over free enzymes.

In this study, a novel co-immobilization biocatalyst for one-pot starch hydrolysis was prepared through shielding enzymes on the Fe3O4/SiO2 core-shell nanospheres by a Fe3+-tannic acid (TA) film. In brief, α-amylase and glucoamylase were covalently immobilized on amino-modified Fe3O4/SiO2 core-shell nanospheres using glutarldehyde as a linker. Then, a Fe3+-TA protective film was formed through the self-assembly of the Fe3+ and TA coordination complex (Fe3+-TA@Fe3O4/SiO2-enzymes). The film acts a "coating" to prevent the enzyme from denaturation and detachment, thus significantly improving its structural and operational stability. Furthermore, the immobilization efficiency reached 90%, and the maximum activity recovery of α-amylase and glucoamylase was 87 and 85%, respectively. More importantly, the bienzyme magnetic nanobiocatalyst with Fe3+-TA film could be simply recovered by a magnet. The Fe3+-TA@Fe3O4/SiO2-enzymes kept 55% of the original activity after reuse for 9 cycles, indicating outstanding reusability. However, the bienzyme magnetic nanobiocatalyst without Fe3+-TA film maintained 28% of the initial activity.

Enzyme immobilization on solid supports is a valuable approach to address enzyme stability and reuse for continuous operations. However, the enzymes immobilized on the external surface of solid supports may not be protected by carriers and suffer inactivation caused by denaturing stresses and hazard external environment. Herein, we describe for the first time a enzyme-shielding strategy to prepare hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of supramolecular metal-organic coordination complex (tannic acid (TA) and Fe3+) at the surface of immobilized catalase on Fe3O4/silica core-shell nanospheres to grow a protective nanocoating (Fe3+-TA nanocoating). The nanocoatings around the immobilized catalase (Fe3+-TA@Fe3O4/SiO2-catalase) provide a “shield effect” to protect from biological, thermal and chemical degradation for enzyme. As a result, the stability of immobilized catalase against proteolytic agent, denaturants and heat were improved remarkably compared to the immobilized catalase without a protective nanocoating and free catalase. More importantly, the recycling of the immobilized catalase was improved remarkably. The Fe3+-TA@Fe3O4/SiO2-catalase still retained 55% of their original activity after 9 cycles, whereas the immobilized catalase without a protective nanocoating only retained 20% of original activity. These results demonstrated that the novel enzyme-shielding strategy is an efficient method to obtain stable and recycled biocatalyst with yolk-shell structure.

In the past few years, organic–inorganic hybrid nanoflowers technology has been emerged as an effective immobilization method. This method has motivated a considerable interest in exploiting them as a potential matrix for biomolecule immobilization due to their simple synthesis, high efficiency, great promise of enhancing biomolecule stability, activity and even selectivity. Recent years, many efforts have focused on this topic to develop biomolecule-inorganic hybrid nanoflowers with potential applications. In this review, recent advances in functional biomolecule-inorganic hybrid nanoflowers are discussed with an emphasis on the novel synthesizing strategies, process optimization, and their potential applications in biosensor, biocatalysis, drug delivery, and chemical analysis. Trends in current developments toward the rational design of these nanoflowers are identified.

We report a novel “anchor–shield” approach for synthesizing a yolk–shell-structured biocatalytic system that consists of a phenylalanine ammonia lyase (PAL) protein particle core and a hollow silica shell with large mesopores by a combination of CaCO3 microtemplates and biomimetic silicification. The method is established upon filling porous CaCO3 cores with PAL via co-precipitation, controlled self-assembly and polycondensation of silanes, cross-link of the PAL molecules, and subsequent CaCO3 dissolution. During this process, the self-assembled layer of cetyltrimethylammonium bromide served as a structure-directing agent of the mesostructure and directed the overgrowth of the mesostructured silica on the external surface of PAL/CaCO3 hybrid microspheres; after CaCO3 dissolution, the cross-linked PAL particles were encapsulated in the hollow silica shell. The hollow silica shell around the enzyme particles provided a “shield” to protect from biological, thermal, and chemical degradation for the enzyme. As...

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising method for enzyme immobilization due to its simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications in some cases. Here, monodisperse spherical CLEAs of phenylalanine ammonia lyase (PAL microspheres) were prepared based on CaCO3 microtemplates. The preparation procedure involves filling porous CaCO3 microtemplates with the protein by salt precipitation, glutaraldehyde crosslinking, and dissolution of the microtemplates. The formulation of CaCO3 templates with controlled size was studied in detail. Characterization of the prepared PAL microspheres was investigated. The results showed that the PAL microspheres with high immobilization efficiency (79%) exhibited excellent stability, including increased tolerance to proteolysis, low pH, and denaturants, and excellent mechanical properties. For example, free PAL almost lost all activity after they were incubated in the presence of trypsin for 2min, whereas PAL microspheres still retained 95% of their initial activity. Moreover, scanning electron microscope, transmission electron microscope, and N2 adsorption-desorption isotherms revealed that the resultant PAL microspheres possessed good monodispersity and mesoporous structure instead of the amorphous clusters of conventional CLEAs with few pores. Compared with conventional CLEAs, the monodisperse PAL microspheres with mesoporous make them more potentially useful for biomedical and biotechnological applications.

BACKGROUND Cross-linking enzyme aggregates (CLEAs) seem to be a promising immobilization method due to mild processing conditions and short preparation time. However, the CLEAs prepared in aqueous solution exhibit amorphous large clusters that cause significant mass-transfer limitations, leading to low catalytic efficiency. In this work, novel spherical CLEAs of lipase from bovine pancreas (spherical CLEAs) were prepared within droplets dispersed in a water-in-hydrophobic ionic liquid microemulsion rather than a conventional aqueous solution. The properties of the spherical CLEAs were determined. RESULTS The resultant CLEAs exhibited spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs, and showed higher activity recovery (84.6%) than that of conventional CLEAs (52.8%). The high activity recovery of spherical CLEAs could be attributed to both interfacial activation of the microemulsion system and the well-defined structure of the spherical CLEAs, which not only activated lipases in an active form, but also decreased mass-transfer limitations. Moreover, the thermostability, storage stability, tolerance against hydrophilic solvents, and mechanical stability of the spherical CLEAs were enhanced significantly. The spherical CLEAs evidenced excellent reusability in comparison with conventional CLEAs. CONCLUSIONS The novel strategy in this study might open an attractive way towards effectively improving the catalytic properties of traditional CLEAs. © 2016 Society of Chemical Industry

Highly stable and easily recyclable hybrid magnetic cross-linked lipase aggregates (HM-CSL-CLEAs) were prepared by coaggregation of lipase aggregates with nonfunctionalized magnetic nanoparticles and subsequent chemical cross-linking with glutaraldehyde. Analysis by SEM and CLSM indicated that the CLEAs were embedded in nanoparticle aggregates instead of covalently immobilized. The resulting HM-CSL-CLEAs exhibited higher thermostability, storage stability, and reusability than standard CLEAs. For example, HM-CSL-CLEAs maintained >60% of their initial activity after 40 min of incubation at 60 °C, whereas standard CLEAs lost most of their activities. The HM-CSL-CLEAs can be easily recovered from the reaction mixture by an external magnetic field. Moreover, the H2O2 tolerance of the lipase in HM-CSL-CLEAs was also enhanced, which could relieve the inhibitory effect on lipase activity. A high conversion yield (55%) for the epoxidation of oleic acid using H2O2 as oxidizing agent was achieved by HM-CSL-CLEAs.

Ultrahigh-vacuum conditions for microelectromechanical systems (MEMS) packaging can be achieved and sustained using a nonevaporable getter (NEG) film technology. Better understanding of the processes involved in film deposition, activation, and poisoning could help optimization and engineering of the film properties. In the present work, highly porous ZrCoCe films were deposited on (100) silicon substrates by direct current magnetron sputtering. Film morphology and performance dependence on deposition pressure, substrate temperature and substrate outgassing were investigated. The studies reveal that the pressure of the Ar sputtering gas affects significantly the film structure. High working pressures produce open columnar structures, whereas low pressures produce dense layers. It is demonstrated that the variation of the substrate temperature results in a significant modification of the film morphology. The highest sorption speed obtained for substrate temperature of 150 °C, is clearly correlated with the high specific surface area and porosity of the ZrCoCe film. It has also been confirmed that sorption properties can be improved by substrate outgassing prior to deposition.

Zeolitic imidazole framework-8 (ZIF-8) with tunable pore sizes and high surface areas have recently emerged as a promising support for immobilizing enzymes. However, the instability in the aqueous acidic environment and difficulty of recovery has limited their practical applications in some cases. In this study, catalase/ZIF-8 composites with a protective nanocoating were prepared by the controlled self-assembly of silanes or coordination complexes (tannic acid (TA) and Fe3+). The properties of the catalase (CAT)/ZIF-8 composites with a protective nanocoating were also determined. The recovered activity of CAT/ZIF-8 and CAT/ZIF-8 with protective nanocoating was 70% and 65%, respectively. Compared with the conventional CAT/ZIF-8 composites, CAT/ZIF-8 with protective nanocoating exhibited excellent acid resistance. For example, after treatment for 60 min in phosphate buffer solution (pH 3.0), CAT/ZIF-8 composites only maintained 20% of their initial activity (about 12 U/mg). However, CAT/ZIF-8 with a protective nanocoating could still retain about 50% of its initial activity (about 10 U/mg). Meanwhile, the thermostability and storage stability of the CAT/ZIF-8 composites was enhanced significantly due to the presence of nanocoating compared with conventional CAT/ZIF-8. More importantly, the CAT/ZIF-8 with a protective nanocoating retained 40% of its initial activity after 7 cycles, whereas CAT/ZIF-8 only retained 8% of the initial activity. The approach in this study could be an efficient strategy to prepare enzyme/ZIF-8 composites with both high acid resistance and excellent recyclability.

In this study, polyvinyl alcohol (PVA)-degrading bacteria were screened from sludge samples using PVA as a sole source of carbon. A novel strain was obtained and identified as Bacillus niacini based on the analysis of a partial 16S rDNA nucleotide sequence and morphological characteristics. PVA-degrading enzyme (PVAase) from Bacillus niacini was immobilized as cross-linked enzyme aggregates (CLEAs) via precipitation with ammonium sulfate followed by glutaraldehyde cross-linking. The effects of precipitation and cross-linking on PVAase-CLEAs activity were investigated and characterized. 70% ammonium sulfate and 1.5% glutaraldehyde were used for precipitation and 1-h cross-linking reaction. The activity recovery of PVAase-CLEAs was approximately 90% starting from free PVAase, suggesting non-purification steps are required for extended use. No significant differences in optimum pH and temperature values of the PVAase were recorded after immobilization. The PVAase-CLEAs showed a ball-like morphology and enhanced PVA degradation efficiency in comparison with the free PVAase in solution. Furthermore, the PVAase-CLEAs exhibited excellent thermal stability, pH stability and storage stability compared to free PVAase. The PVAase-CLEAs retained about 75% of initial PVAase activity after 4 cycles of use. These results suggest that this CLEA is potentially usable for PVA degradation in industrial applications.

In the past few decades, the enzyme immobilization technology has been exploited a lot and thus became a matter of rational design. Immobilization is an alternative approach to bio-catalysis with the added benefits, adaptability to automation and high-throughput applications. Immobilization-based approaches represent simple but effective routes for engineering enzyme catalysts with higher activities than wild-type or pristine counterparts. From the chemistry viewpoint, the concept of stabilization via manipulation of functional entities, the enzyme surfaces have been an important driving force for immobilizing purposes. In addition, the unique physiochemical and structural functionalities of pristine or engineered cues, or insoluble support matrices (carrier) such as mean particle diameter, swelling behavior, mechanical strength, and compression behavior are of supreme interest and importance for the performance of the immobilized systems. Immobilization of peroxidases into/onto insoluble support matrices is advantageous for practical applications due to convenience in handling, ease separation of enzymes from a reaction mixture and the reusability. A plethora of literature is available explaining individual immobilization system. However, current literature lacks the chemistry viewpoint of immobilization. This review work presents state-of-the-art "Smart" chemistry of immobilization and novel potentialities of several materials-based cues with different geometries including microspheres, hydrogels and polymeric membranes, nanoparticles, nanofibers, composite and hybrid or blended support materials. The involvement of various functional groups including amino, thiol, carboxylic, hydroxyl, and epoxy groups via "click" chemistry, amine chemistry, thiol chemistry, carboxyl chemistry, and epoxy chemistry over the protein surfaces is discussed.

BACKGROUND Encapsulation of enzymes in biomimetic silica seems to be a promising immobilization method due to mild processing conditions and short preparation time. However, biomimetic silica particles may be considered too small for industrial applications because they result in difficult recovery. Furthermore, the use of R5 peptide or polymers as catalyst usually causes protein denaturation. To overcome these drawbacks, a new stable and recyclable biocatalyst based on co-encapsulation of magnetic nanoparticles and phenylalanine ammonia lyase (PAL) in biomimetic silica has been prepared using enzyme-assisted direct condensation reactions of silicon oxide with tetramethoxysilane (TMOS). RESULTS Under optimal conditions (10 mg mL−1 nanoparticles, 4.5 U mL−1 PAL, 0.8 mol L−1 TMOS), the highest activity recovery (52%) of encapsulated PAL was obtained. Compared with conventional PAL encapsulated in biomimetic silica (28%), the activity recovery was increased 1.86-fold. TEM, CLSM, magnetisation measurements, and FTIR confirmed that the magnetic nanopaticles and PAL had been co-encapsulated into the biomimetic silica. The resulting immobilized PAL exhibited higher activity and stability than native PAL, and the immobilized PAL can be easily separated by applying a magnetic field. CONCLUSIONS This work provides a convenient strategy to prepare stable and easy recycled biocatalyst with excellent catalytic performance. © 2015 Society of Chemical Industry

Metal-organic frameworks (MOFs) have recently emerged as a promising candidates for the immobilization of enzymes due to their diversified structures and porosity. However, a lack of good size and morphological control over the as-prepared MOFs has limited their practical applications in some cases. Herein, instead of zeolitic imidazolate framework-8 (ZIF-8) with the standard rhombic dodecahedral morphology, we successfully synthesize a novel mesoporous catalase@ZIF composite with cruciate flower-like morphology by embedding catalase molecules into uniformly sized ZIF crystals. With extraordinarily large mesopore size and high protein loading capacity, the catalase@ZIF composites with cruciate flower-like morphology exhibit 400% higher activity than that of catalase@ZIF composites with conventional rhombic dodecahedral morphology, and show higher reusability than conventional rhombic dodecahedral morphology. More importantly, we demonstrate for the first time that the biomacromolecules (proteins) can not directly regulate the crystal size, morphology, and crystallinity of ZIF-8. Moreover, the crystal morphology of ZIF has primary dependence on concentrations of 2-methylimidazole and Zn

With the aim of providing a highly stable and easy recyclable biocatalyst, we prepared a new biocatalyst formulation based on cross-linked enzyme aggregate (CLEA) technology by the physical adsorption of cross-linked phenylalanine ammonia lyase on a highly porous microspherical silica gel solid support (S-CLEAs). The main factors in the preparation of S-CLEAs were optimized by response surface methodology (RSM). Analysis by scanning electron microscopy (SEM), FT-IR spectroscopy, and confocal laser scanning microscopy (CLSM) indicated that the CLEAs were successfully immobilized on the crude-pored microspherical silica gel. Compared to standard CLEAs, the S-CLEAs can be easily separated from the reaction mixture by natural precipitation without centrifugation and filtration treatments. Although the S-CLEAs revealed an excellent stability of the enzyme against high temperature and denaturants, the volumetric activity of the S-CLEAs decreased because of the presence of silica gel. In a word, the S-CLEA technology in this study might open an attractive route toward effectively improving the catalytic properties of traditional CLEAs.

The aim of this study was to develop an environmentally safe and efficient method for phenyllactic acid (PLA) production using whole-cell cascade catalysis with l -amino acid deaminase ( l -AAD), lactate dehydrogenase (LDH), and formate dehydrogenase (FDH). The PPA titer was low due to relatively low expression of LDH, intermediate accumulation, and lack of cofactors. To address this issue, ribosome binding site regulation, gene duplication, and induction optimization were performed to increased the PLA titer to 43.8 g/L. Then co-substrates (glucose, yeast extract, and glycerol) were used to increase NADH concentration and cell stability, resulting that the PLA titer was increased to 54.0 g/L, which is the highest reported production by biocatalyst. Finally, glucose was replaced with wheat straw hydrolysate as co-substrate to decrease the cost. Notably, the strategies reported herein may be generally applicable to other whole-cell cascade biocatalysts.

Cross-linked enzyme aggregates (CLEAs) have recently emerged as a promising tool for enzyme immobilization because of their simplicity and low cost. However, a lack of good size and morphological control over the as-prepared CLEAs has limited their practical applications. For example, the prepared CLEAs exhibit amorphous large clusters that would cause significant mass-transfer limitations, which lead to a low catalytic efficiency. Here, inspired by biomineralized core-shell structures in nature, we develop a novel mesoporous spherical CLEA with a biosilica shell by using phenylalanine ammonia lyase based on CaCO3 microtemplates and biomimetic mineralization. The resultant CLEAs exhibited a spherical structure with good monodispersity instead of the amorphous clusters of conventional CLEAs and showed activity higher than that of conventional CLEAs. Moreover, the thermostability, tolerance against denaturants, and mechanical stability of the spherical CLEAs with a biosilica shell were enhanced significantly compared with those of conventional CLEAs. In particular, the spherical CLEAs with a biosilica shell retained 70% of their original activity after 13 cycles, whereas the conventional CLEAs retained only 35% of their original activity. This approach could be an efficient strategy for improving the catalytic properties of CLEAs.

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel.

Aims: To improve exopolysaccharides (EPS) production of Cordyceps militaris (C. militaris), effects of different culture method on mycelial biomass and EPS production in the submerged culture of C. militaris were investigated. Methods and Results: A new two-stage fermentation process for EPS production of C. militaris was designed in this work. Central composite design (CCD) was utilized to optimize the two-stage fermentation process. The results showed that the two-stage fermentation process for EPS production was superior to other culture method (conventional static culture and shake culture). CCD revealed that the optimum values of the test variables for EPS production were shaken for 140 h followed by 130-h static culture. The maximum EPS production reached 3AE 2gl )1 under optimized two-stage culture and was about 2AE3fold and 1AE6-fold in comparison with those of original static culture and shake culture. Conclusions: It was indicated that a new two-stage culture method obtained in this work possessed a high potential for the industrial production for EPS of C. militaris. Significance and Impact of the Study: The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of C. militaris for the production of bioactive metabolites.

Statistical experimental design strategy (SES) was applied to optimize the medium for the exopolysaccharides (EPS) production of Cordyceps militaris by submerged culture in shaker flask. A significant influence of the glucose and peptone on the EPS production was first evaluated by using a Plackett-Buman design. Then, steepest ascent method was employed to approach the experimental design space. Last, these factors were further optimized using central composite design (CCD) and response surface methodology (RSM). A quadratic model was found to fit the EPS production. The optimum values of the tested variables for the production of EPS were 48.67 g/L glucose, 12.56 g/L peptone, 1 g/L KH2PO4, 10 g/L yeast extract, and 0.5 g/L MgSO4·7H2O. Under optimization of culture conditions, the EPS production was enhanced from 0.78 to 1.96 g/L. In comparison with that of original culture conditions, 2.5 fold increase was obtained.

To develop a new method for producing aspartame (APM), a simple and efficient strategy for the isolation of certain microorganisms producing APM from soil samples was designed. A newly strain with secreting certain specific dipeptidase to directly synthesize APM from the substrates of l-aspartic acid (l-Asp) and l-phenylalanine methyl ester (l-PM) without protection for amino acid side chains was screened from soil samples. APM concentration in reaction mixture was quantified by high performance liquid chromatography (HPLC), yield reached 0.015 g/L. Examination of the general morphological characteristics and data from the sequence analysis of the rDNA-ITS gene led to identification of the isolate as a strain of fungal endophyte spp. The newly isolated strain had a high potential for application in industrial processes for APM production. In particular, this new method was low cost for synthesis of APM during the reaction due to avoiding protection for amino acid side chains and optical resolution of the mixtures. As we known it, this is first report that a newly strain with a high potential for selective synthesis of the APM was isolated from soil.

Some approaches for improving recombinant phenylalanine ammonia lyase (PAL) stability in Escherichia coli during the enzymatic methods of l-phenylalanine (l-Phe) production were developed following preliminary studies by means of statistical-based experiment designs (response surface method). The traditional non-statistical technology was used to screen four critical factors for PAL stability during the bioconversion process, viz., glycerin, sucrose, 1,4-dithiothreitol (DTT), and MgSO(4). The central composite design (CCD) was applied to optimize the combined effect of critical factors for recombinant PAL stability and understand the relationship between the factors and PAL stability. The optimum values for testing variables were 13.04 mM glycerin, 1.87 mM sucrose, 4.09 mM DTT, and 69 mM Mg(2+). A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory because the coefficient of determination was 0.88. The maximum PAL activity was retained as 67.73 units/g after three successive cycles of bioconversion. In comparison to initial PAL activity, the loss of PAL activity was only 22%. PAL activity was enhanced about 23% in comparison to the control (without any stabilizer additives). PAL stability was significantly improved during successive bioconversion. The results obtained here verified the effectiveness of the applied methodology and may be helpful for l-Phe production on an industrial scale.

Ba0.6Sr0.4Ti1−xMnxO3 [BST(Mn), x = 0.0000, 0.0025, 0.0050, 0.0075, 0.0100 and 0.0125] powders were synthesized by solid-state reaction process, and then BST(Mn)/MgO ceramic composites (60 wt% MgO) were fabricated by conventional ceramic process. Mn was found distributed in the BST grains homogeneously after sintering at 1420 °C for 4 h, neither grain-boundary segregation nor diffusion into the MgO grains could be found obviously. The dielectric nonlinearity of the materials was enhanced with the addition of Mn-dopant, and relatively high tunability, more than 27% (8 kV/mm DC field, 500 Hz), was obtained in BST(Mn)/MgO samples with x ≤ 0.0100. The dielectric loss at microwave frequency of the samples with Mn-dopant was significantly reduced, and a tremendously low dielectric loss was acquired for the sample with x = 0.0050, which is only 4.5 × 10−3 (at 3.64 GHz). Both the low dielectric loss and enhanced tunable properties of the BST(Mn)/MgO composites are useful for tunable microwave applications.

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH4)2HPO4 and MgSO4. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH4)2HPO4 7.02 g/L and MgSO4 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.

The effects of moderate pressure (0.1-2.5 MPa) on viability, cell membrane permeability and catalyzing activity of Absidia coerulea for RSA were investigated. A new method for improving the production of Hydrocorti- sone (HC) from 17α-hydroxypregn-4-en-3, 20-dione-21-acetate by Absidia coerulea in moderate pressure was devel- oped. The results showed that the morphology of Abasidia coerulea mycelium was changed in moderate pressure, Absidia coerulea mycelium seemed to be loosed, and cell membrane permeability of Abasidia coerulea mycelium was improved. However, the viability of Abasidia coerulea mycelium could keep high level. Moreover, the yield of HC was improved over 1.25-fold as compared with that of the control (untreated cells), to give the yield of HC as 350 mg/l, when the Abasidia coerulea mycelium was treated with 0.5 Mpa the atmosphere as the pressure media. Especially, the production of HC with atmosphere as the pressure media (0.5 MPa) could be increased by the addition of H2O2 (60 mmol/l); the relative yield of HC in moderate pressure was enriched by over 4.5% in comparison with the control. The major com- position of bioconverted mixture was reduced. It was indicated that the new approach (moderate pressure) obtained in this work possessed a high potential for the industrial production of HC.

With CO2 and N2 as the pressure media, the effects of the moderate pressure (0.1–1.0MPa) and the holding time on the conductivities of the cell suspension of Saccharomyces cerevisiae CICC1447 and Saccharomyces cerevisiae CICC1339, as well as the absorbances of the supernatant (after centrifuged) at 280 nm (A280) and 260 nm (A260) were determined. The membrane permeability of Saccharomyces cerevisiae CICC1447 increased significantly and the cell leakage was aggravated with the pressure increase. For Saccharomyces cerevisiae CICC1339, the conductivity of the cell suspension, A280 and A260 of the supernatant fluctuated with the pressure increase; as a whole, they increased with pressure. Different from high pressure, a moderate pressure not only remarkably improved the permeability of the yeast cell membrane, but also kept yeast cell viability; moreover, the integrity of the yeast cell membrane could be maintained.

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal com- position of the batch medium was 2% glucose, 1% yeast extract, 0.7% K2HPO4, 0.8% KH2PO4, 0.5% (NH4)2SO4, 0.1% MgSO4·7H2O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37 o C to 42 o C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD600 about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.

Aim: To improve phenylalanine ammonia lyase (E.C.4·3·1·5-PAL) activity in recombinant Escherichia coli. Some methods for enrichment of PAL activity in recombinant E. coli JM109 were described. In an effort to create a rich enzyme source these methods would lead to improvements in the production of l-phenylalanine. Methods and Results: The possibilities of enriching PAL activity in recombinant E. coli was investigated by using individual and combinations of amino acids, organic solvents and surfactants. PAL activity was induced by adding combination of l-phenylalanine and l-tyrosine, activities as high as 64·3 U g−1of cells were obtained and enzyme activity was enriched by over 3·5-fold in comparison with the control. Permeabilization with cetyl trimethyl ammonium bromide or the acetone significantly enriched cellular PAL activity, which improved over 8·2- and 9·0-fold compared with the control, as high as 148·5 and 164·5 U g−1of cells respectively. Conclusion: These efforts may provide some effective methods for enhancing l-phenylalanine ammonia lyase activity. Significance and Impact of the Study: These approaches for manipulating recombinant E. coli in an effort to create a rich enzyme source would serve as a biotechnologically important protocol for production of l-phenylalanine.