Improved Enzyme Activity Research Articles

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water-soluble IL 1-ethyl-3-methylimidazolium ethylsulfate ([emim][EtSO4 ]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl-agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis-Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO4 ]. When concentration of the IL was augmented, the values of Vmax for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase-glyoxyl-agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO4 ]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first-order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO4 ]. Finally, thermal stability was scarcely affected by the presence of the IL.

Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons

A novel electrochemical microbial biosensor for the rapid monitoring of p-nitrophenyl-substituted organophosphates (OPs) compounds based on glass carbon electrode (GCE) modified with both ordered mesopore carbons (OMCs) and cell surface-expressed organophosphorus hydrolase (OPH) (OPH-bacteria/OMCs/GCE) was described in this paper. The genetically engineered Escherichia coli strain surface displayed mutant OPH (S5) with improved enzyme activity and favorable stability was constructed using a newly identified N-terminal of ice nucleation protein as an anchoring motif, which can be used directly without further time-consuming enzyme-extraction and purification, thereafter greatly improved the stability of the enzyme. Compared to OPH-bacteria modified GCE (OPH-bacteria/GCE), the OPH-bacteria/OMCs/GCE not only significantly enhanced the current response but also reduced the oxidation overpotential towards oxidizable p-nitrophenol (p-NP), which was the hydrolysate of p-nitrophenyl-substituted OPs. Under the optimized experimental conditions, at +0.84V (vs. SCE), the current–time curve was performed with varying OPs concentration. The current response was linear with paraoxon concentration within 0.05–25μM. Similarly, linear range of 0.05–25μM was found for parathion, and 0.08–30μM for methyl parathion. The low limits of detection were evaluated to be 9.0nM for paraoxon, 10nM for parathion and 15nM for methyl parathion (S/N=3). Thus, a highly specific, sensitive and rapid microbial biosensor was established, which holds great promise for on-site detection of trace p-nitrophenyl-substituted OPs.

Dl-2 Aminobutyric acid and calliterpinone are the potential stimulators of Trichoderma cellulase activities

Trichoderma citrinoviride EB-104 has recently been developed as a hyper β-glucosidase producing mutant strain. In the present study, efforts were made to further enhance its enzyme titers using low molecular weight stimulators viz., calliterpinone (a natural plant diterpenoid), dl-2-aminobutyric acid (isomer of an amino acid, aminobutyric acid) and jasmonic acid (a natural plant growth regulator). Among test stimulators, calliterpinone and dl-2-aminobutyric acid significantly enhanced activities of all three enzymes of cellulase complex i.e., FPase, endoglucanase and β-glucosidase. A 5.0 and 2.7 fold increase in FPase and endoglucanase activities, respectively, could be achieved by incorporating 12.5 mmol dl-2-aminobutyric acid into fermentation media. On the other hand, more than fivefold enhanced β-glucosidase activity was evident with 10 μmol of calliterpinone, raising its activity from 8.28 to 43.38 IU cm−3. This β-glucosidase activity of T. citrinoviride EB-104 is far higher than that of reported earlier. These test stimulators, which improved enzyme activities of cellulase complex in T. citrinoviride EB-104, may also be explored for enhancing the activities of other fungal enzymes.

Formation of disulfide bonds in insect prophenoloxidase enhances immunity through improving enzyme activity and stability

Type 3 copper proteins, including insect prophenoloxidase (PPO), contain two copper atoms in the active site pocket and can oxidize phenols. Insect PPO plays an important role in immunity. Insects and other invertebrates show limited recovery from pathogen invasion and wounds if phenoloxidase (PO) activity is low. In most insect PPOs, two disulfide bonds are present near the C-terminus. However, in Pimpla hypochondriaca (a parasitoid wasp), each PPO contains one disulfide bond. We thus questioned whether the formation of two sulfide bonds in insect PPOs improved protein stability and/or increased insect innate immunity over time. Using Drosophila melanogaster PPO1 as a model, one or two disulfide bonds were deleted to evaluate the importance of disulfide bonds in insect immunity. rPPO1 and mutants lacking disulfide bonds could be expressed and showed PO activity. However, the PO activities of mutants lacking one or two disulfide bonds significantly decreased. Deletion of disulfide bonds also reduced PPO thermostability. Furthermore, antibacterial activities against Escherichia coli and Bacillus subtilis significantly decreased when disulfide bonds were deleted. Therefore, the formation of two disulfide bond(s) in insect PPO enhances antibacterial activity by increasing PO activity and stability.

CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (kd), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.

The bacterial Entner–Doudoroff pathway does not replace glycolysis in Saccharomyces cerevisiae due to the lack of activity of iron–sulfur cluster enzyme 6-phosphogluconate dehydratase

Replacement of the glycolytic pathway of Saccharomyces cerevisiae by a bacterial Entner–Doudoroff pathway (EDP) would result in lower ATP production and therefore a lower biomass yield is expected that would further allow higher products yields in the fermentation of sugars. To establish catabolism of glucose via the EDP in S. cerevisiae requires expression of only two additional enzyme activities, 6-phosphogluconate dehydratase (PGDH) and KDPG aldolase. In this work, KDPG aldolase from Escherichia coli could be successfully expressed in the yeast cytosol with very high enzyme activity. Nevertheless, simultaneous expression of KDPG aldolase and a codon optimized PGDH gene of E. coli could not replace glycolysis or the pentose phosphate pathway in growth experiments. It could be shown that this was due to the very low enzyme activity of PGDH. This bacterial enzyme is a [4Fe–4S] iron–sulfur cluster protein. Several attempts to improve the availability of iron–sulfur clusters or iron in the yeast cells, to attract the iron–sulfur cluster assembly machinery to Leu1-PGDH fusion proteins or to localize the PGDH in the mitochondria did not result in improved enzyme activities. From our results we conclude that establishing functional expression of iron–sulfur cluster enzymes will be a major task for the integration of the EDP and other biochemical pathways in yeast.

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18-10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.

Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat/KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat/KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).

Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium

BackgroundBurkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium.ResultsIn this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterification of 1-phenylethanol with vinyl acetate was chosen as model reaction. Compared with its free form, the enzyme activity and enantioselectivity (ees) of the immobilized lipase have been significantly enhanced. The immobilized BCL exhibited a satisfactory thermostability over a wide range of temperature (from 10 to 65°C) and an excellent catalytic efficiency. After being used for more than 30 successive batches, the immobilized lipase still kept most of its activity. In comparison with other immobilized lipases, the immobilized BCL also exhibits better catalytic efficiency, which indicates a significant potential in industrial applications.ConclusionThe results of this study have proved that MPR-NKA was an excellent support for immobilization of lipase via the methods of N2 adsorption–desorption, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform-infrared spectroscopy (FT-IR). The improvement of enzyme activity and ees for the immobilized lipase was closely correlated with the alteration of its secondary structure. This information may contribute to a better understanding of the mechanism of immobilization and enzymatic biotransformation in non-aqueous medium.

Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16U/OD600 using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55°C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5–150μM paraoxon, 1–200μM parathion and 2.5–200μM methyl parathion, and limits of detection were 0.2μM, 0.4μM and 1μM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.

ChemInform Abstract: Modifying Enzyme Activity and Selectivity by Immobilization

AbstractReview: 61 refs.

Fructooligosacharides production in aqueous medium with inulinase from Aspergillus niger and Kluyveromyces marxianus NRRL Y-7571 immobilized and treated in pressurized CO2

This work investigated the influence of compressed CO2 treatment on the enzymatic activity of immobilized inulinases, and the production of fructooligosacharides in aqueous medium using these enzymes. The effects of system pressure, exposure time and depressurization rate on the enzymatic activity were evaluated through central composite designs (CCD) 23. Inulinase from Kluyveromyces marxianus NRRL Y-7571 presented an increase of 104% in the residual activity using CO2 at 275bar submitted to 6h treatment, at a depressurization rate of 10kgm−3min−1. For Aspergillus niger commercial inulinase, a decrease in enzyme activity was observed (residual activity of 39%) using CO2 treatment at 75bar for 6h exposure at the highest depressurization rate (200kgm−3min−1). Enzymatic activities changed significantly depending on the enzyme source and the experimental treatment conditions investigated. The values of FOS obtained using inulinases from A. niger were 30.64% of GF2; 13.90% of GF3 and 2.88% of GF4 in the medium containing inulin as substrate. Results demonstrate that the use of compressed CO2 might be of technological importance as a preceding, preparation step, to improve enzyme activity, hence helping the development of new biotransformation processes.

Proteomic analysis of F1F0-ATP synthase super-assembly in mitochondria of cardiomyoblasts undergoing differentiation to the cardiac lineage

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. An increasing number of data highlighted their role for cellular differentiation processes. We investigated differences in ATP synthase supra-molecular organization occurring in H9c2 cardiomyoblasts in the course of cardiac-like differentiation, along with ATP synthase biogenesis and maturation of mitochondrial cristae morphology. Using BN-PAGE analysis combined with one-step mild detergent extraction from mitochondria, a significant increase in dimer/monomer ratio was observed, indicating a distinct rise in the stability of the enzyme super-assembly. Remarkably, sub-stoichiometric mean values for ATP synthase subunit e were determined in both parental and cardiac-like H9c2 by an MS-based quantitative proteomics approach. This indicates a similar high proportion of complex molecules lacking subunit e in both cell types, and suggests a minor contribution of this component in the observed changes. 2D BN-PAGE/immunoblotting analysis and MS/MS analysis on single BN-PAGE band showed that the amount of inhibitor protein IF1 bound within the ATP synthase complexes increased in cardiac-like H9c2 and appeared greater in the dimer. In concomitance, a consistent improvement of enzyme activity, measured as both ATP synthesis and ATP hydrolysis rate, was observed, despite the increase of bound IF1 evocative of a greater inhibitory effect on the enzyme ATPase activity. The results suggest i) a role for IF1 in promoting dimer stabilization and super-assembly in H9c2 with physiological IF1 expression levels, likely unveiled by the fact that the contacts through accessory subunit e appear to be partially destabilized, ii) a link between dimer stabilization and enzyme activation.

A Review of the Role of Amphiphiles in Biomass to Ethanol Conversion

One of the concerns for economical production of ethanol from biomass is the large volume and high cost of the cellulolytic enzymes used to convert biomass into fermentable sugars. The presence of acetyl groups in hemicellulose and lignin in plant cell walls reduces accessibility of biomass to the enzymes and makes conversion a slow process. In addition to low enzyme accessibility, a rapid deactivation of cellulases during biomass hydrolysis can be another factor contributing to the low sugar recovery. As of now, the economical reduction in lignin content of the biomass is considered a bottleneck, and raises issues for several reasons. The presence of lignin in biomass reduces the swelling of cellulose fibrils and accessibility of enzyme to carbohydrate polymers. It also causes an irreversible adsorption of the cellulolytic enzymes that prevents effective enzyme activity and recycling. Amphiphiles, such as surfactants and proteins have been found to improve enzyme activity by several mechanisms of action that are not yet fully understood. Reduction in irreversible adsorption of enzyme to non-specific sites, reduction in viscosity of liquid and surface tension and consequently reduced contact of enzyme with air-liquid interface, and modifications in biomass chemical structure are some of the benefits derived from surface active molecules. Application of some of these amphiphiles could potentially reduce the capital and operating costs of bioethanol production by reducing fermentation time and the amount of enzyme used for saccharification of biomass. In this review article, the benefit of applying amphiphiles at various stages of ethanol production (i.e., pretreatment, hydrolysis and hydrolysis-fermentation) is reviewed and the proposed mechanisms of actions are described.

Sodium Dodecyl Sulphate, a Strong Inducer of Thermostable Glucanhydrolase Secretion from a Derepressed Mutant Strain of Bacillus alcalophilus GCBNA-4

In the present study, we report the optimisation of batch conditions for improved α-1,4-glucan-glucanohydrolase (GGH) secretion by a nitrous acid (NA)-treated Bacillus alcalophilus. The wild (isolate GCB-18) and NA-derivative (mutant GCBNA-4) were grown in a medium containing 10 g/L nutrient broth, 10 g/L starch, 5 g/L lactose, 2 g/L ammonium sulphate, 2 g/L CaCl2 and phosphate buffer (pH 7.6). Sodium dodecyl sulphate (SDS) was used as an enzyme inducer while batch fermentations were carried out at 40 °C. The mutant produced GGH in 40 h which was 15-fold higher than the wild in presence of SDS. Thermodynamic studies revealed that the mutant culture exhibited the capability for improved enzyme activity over a broad range of temperature (35-70 °C). The enzyme was purified by cation-exchange column chromatography with ~80 % recovery. The performance of fuzzy-logic system control was found to be highly promising for the improved substrate conversion rate. The correlation (1.045E + 0025) among variables demonstrated the model terms as highly significant indicating commercial utility of the culture used (P < 0.05).

Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only <5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Modifying enzyme activity and selectivity by immobilization

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Mutations in the substrate entrance region of -glucosidase from Trichoderma reesei improve enzyme activity and thermostability

β-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave β-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei β-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced k(cat)/K(m) and k(cat) values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-β-D-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei β-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.

Pilot-Scale Gas Double-Dynamic Solid-State Fermentation for the Production of Industrial Enzymes

Presently, the modern solid-state fermentation (SSF) industry remains traditional; there are few designs available for bioreactors operating in SSF on a large scale, which is mainly caused by the problems in engineering that appear when scaling up this technique. Gas double-dynamic solid-state fermentation (GDD-SSF) technology has been proven available in industrial production of biopesticide. However, there are few other examples using the GDD-SSF technology in other fermentation industries. To test its applicability in other fermentation industries, the self-made pilot-scale bioreactor for GDD-SSF of 800 L was used for the production of various typical enzymes. The comparing effects of liquid-solid ratio and air pressure on enzyme production in different fermentation modes were investigated. Results showed that GDD-SSF offered greater advantages to the static SSF in industrial enzyme preparation, such as pectinase, glucoamylase, protease, and cellulase. It could not only improve enzyme activities with a largest increase reached 2.84 times but also shorten the fermentation period. Furthermore, decreased temperature gradient was found in GDD-SSF, which is crucial for online controlling in industry production. Thus, the necessary and successful pilot-scale fermentation of various enzymes was a rational indication of applicability to large-scale SSF by GDD-SSF.

Improvement of Cellulase Activity Using Error-Prone Rolling Circle Amplification and Site-Directed Mutagenesis

Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM MnCl2. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.