Optimum Substrate Concentration Research Articles

Improved reuse and affinity of enzyme using immobilized amylase on alginate matrix

Enzyme immobilizations were widely used to increase their shelf life which is essential for the world’s industries. Therefore, amylase immobilized using Na-alginate as a matrix is necessary optimized and characterized. The parameters measured in the optimization of immobilization are the determination of the concentration of sodium alginate and contact time. Characterizations were conducted to determine the optimum concentration of substrate, the value Vmax, Michaelis-Menten constant (KM), pH, temperature, incubation time, and test reuse. The process of immobilized amylase activity test was performed in a continuous flow system using a reactor, and its sugar levels were determined using the Dinitro Salisilat Method (DNS). The results reveal that the immobilized amylase commercial has optimum concentration of Na-alginate of 5% (w/v) and contact time of 90 minutes with an immobilization efficiency value of 43.02%. Furthermore, the immobilized amylase has optimum activity at substrate concentrations of 3.5% (w/v), pH 4, incubation temperature of 40 °C, and a reaction time of 20 minutes with the value of the activity of 2760.4 U / mL. KM value of free amylase and immobilized amylase row are 0.18 mM and 0.15 mM, repectively. The value of KM immobilized amylase is smaller than the free enzyme. It proves that the immobilized amylase has a high affinity for the substrate. The immobilized amylase can be used up to 12 times with a value of the residual activity of 56.7%.

Evaluation of hydrogen yield potential from Chlorella by photo-fermentation under diverse substrate concentration and enzyme loading

Chlorella is widely distributed, can be cultured in waste water and had short growth cycle. The high carbohydrate composition shows great potential for bioenergy output. In this work, concentrated Chlorella solution was adopted as raw material. Reducing sugar concentration, pH, and cumulative bio-hydrogen yield were taken as indexes, the effects of substrate concentration and enzyme (cellulase or neutral protease) load on photo-fermentation bio-hydrogen production process from microalgae biomass were investigated. Results showed that highest cumulative hydrogen yield was obtained at the optimal substrate concentration of 25 g/L, when the load of cellulase and protease are both 15%, the effect is the best which were 16.65 mL, 29.44 mL, and 43.62 mL, respectively. Results fitted well to the Gompertz model, indicating the feasibility of photo-fermentative bio-hydrogen production from concentrated Chlorella.

Covalent immobilization of thioglucosidase from radish seeds for continuous preparation of sulforaphene

As the hydrolysis product of radish thioglucoside catalyzed by thioglucosidase, sulforaphene (SFE) is a kind of functional ingredient with broad market prospects in virtue of its strong anti-oxidation, anti cell mutation, anti-cancer, antibacterial and herbicidal activities. Due to the fact that free thioglucosidase can be only used once, researching immobilized thioglucosidase and its enzymatic properties has determinant impact on the practical preparation of SFE. However, current immobilized thioglucosidase unexceptionally suffer from serious limitations of low mechanical strength and decomposition by microorganisms, not enough to support the enzyme to achieve continuous industrial reaction. In this study, thioglucosidase was separated and purified from radish seeds by means of extraction, precipitation and membrane separation, so the specific activity of thioglucosidase was increased to 8 times of the original. Then the purified thioglucosidase was covalently immobilized on amine-based resin by glutaraldehyde and tests have been performed on the determination of optimal cross-linking time, optimal cross-linking pH and the molar ratio of free enzyme, glutaraldehyde and carrier amino groups. Therewith the immobilized thioglucosidase was proved to possess superior enzymatic properties including pH adaptation, temperature adaptation, thermal stability, reusability and storage stability, compared with the free thioglucosidase. After the optimal substrate concentration and flow rate was found, the yield of SFE produced by the immobilized thioglucosidase reactor column reached 9.10%, and the benefit of immobilized thioglucosidase is 2.91 times that of direct fermentation. The immobilized thioglucosidase on amine-based resin can be further implemented for the production of SFE through enzymatic method instead of chemical synthesis or direct fermentation.

Kinetic of 4-Chlorophenols Biodegradation by Arthrobacter Chlorophenolicus A6

Chlorophenols are listed as priority pollutants both by European community and US EPA. Biodegradation of pchlorophenol (4-CP) was investigated in batch shake flasks by Arthrobacter chlorophenolicus A6 at initial 4-CP concentrations between 25 to 350 mgl-1. The rate of 4-CP removal decreased with increasing initial 4-CP concentrations due to toxic effects on the microorganisms. The growth and biodegradation kinetic of the culture was evaluated. The batch growth profile of the A chlorophenolicus A6 followed substrate inhibition kinetics with the estimated biokinetic parameters of Ksi = 272 mgl-1, Ks = 65 mgl-1 for 4-CP respectively. High inhibition constant (KSI = 272 mg -l) with a KSI/KS ratio of 4.18 indicates superior 4-CP biodegradation potential of the A chlorophenolicus A6. The maximum rate of 4-CP degradation has been achieved at an optimum substrate concentration of Smax = (KSKSI)1/2 = (65×272)1/2 = 133 mgl−1.

Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206).

Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206). 
 
 http://dx.doi.org/10.25130/tjps.24.2019.130

Enzymatic Bioconversion of Cycloastragenol-6-O-β-D-glucoside into Cycloastragenol by a Novel Recombinant β-Glucosidase from Phycicoccus sp. Soil748

ABSTRACT Cycloastragenol (CA), the genuine sapogenin of astragaloside from Astragalus membranaceus, exhibits diverse pharmaceutical activities. Recently, the efficient production of CA has received considerable attention due to rapidly increasing market demands. In this study, enzyme mining was conducted, based on skeleton and glycosyl similarity, to explore an efficient β-glucosidase for CA preparation. A novel β-glucosidase from Phycicoccus sp. Soil748 (Bgps) was discovered, possessing the efficient conversion rate for cycloastragenol-6-O-β-D-glucoside (CMG) into CA. The optimum temperature and pH value of Bgps were determined as 45 °C and 7.0. The results of kinetic analysis suggested that Bgps catalyzed deglycosylation of CMG more efficiently than other substrates. Furthermore, the optimal substrate concentration of Bgps was up to 80 mg/mL with the conversion rate as 99.2%, suggesting its potential application in CA industrial production by biotransformation.

A hazard to human health - pesticide residues in some vegetal and animal foodstuff

Highly efficient asymmetric reduction of 2-octanone to (R)-2-octanol catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells was achieved in a biphasic system. Bioreduction conducted in aqueous single phase buffer was limited due to poor solubility and toxicity towards cells cause by product accumulation. Introduction of [C4MIM]·Ac accelerated the biotransformation process, giving 99% yield, >99% product e.e. and 1.42-fold higher initial reaction rate in conversion of 10 mM 2-octanone as substrate, compared with 99% yield, 97.3% product e.e. and 1.57 μmol min−1 of initial reaction rate in a aqueous single phase buffer system containing 6 mM 2-octanone as the optimal substrate concentration. Moreover, in the [C4MIM]·Ac-containing buffer/n-tetradecane biphasic system, the optimal substrate concentration was enhanced by 83 times (500 mM) in comparison with that in aqueous single phase buffer, resulting in 53.4% yield (267 mM), 99% product e.e. with 8.9 mM g-1 h-1 space time yield.

Kinetic Studies of Na+/K+-ATPase in Tissue Aerobic Thyroid Patients

Na+/K+-ATPase is a prevalent enzyme that maintains the Na+ and K+ gradients across the cell membrane by transporting three Na+ out and two K+ into the cell, the aim of this study is to provide detailed mechanistic insights, potentially with important effects on physiological regulation of active Na and K transport in tissues of Aerobic Thyroid Patient. Thyroid tissues were obtained from a 35 year old patients, the operation was carried out at the Al-Hadi Specialist Hospital in Samarra city, the sample was stored at -20ºC until used. The purification protocol included Salt Precipitation, Ion Exchange Chromatography, Gel Filtration and Electrophoresis, a spectrophotometric method was used to determine the enzyme activity. kinetic parameters was also obtained for the enzyme. Partial purification of Na+/K+-ATPase revealed two isoenzymes (I ,II). The purity of separated isoenzymes were proved by SDS-PAGE electrophoresis. The kinetic characteristics of Na+/K+-ATPase showed that optimum substrate concentration about 1.5mM, Km 1.052mM, and Vmax 6.062, optimum temperature was 37 ºC, optimum pH 7.4 and optimum time in 25 min. Na+/K+-ATPase purified from Thyroid tissue has distinct kinetic characteristic that reflects the importance of intracellular regulation of specific Na+/K+-ATPase pump which gives cells the ability to precisely coordinate to their physiological requirements .

Laccase: addressing the ambivalence associated with the calculation of enzyme activity.

Laccase (benzenediol: oxygen oxidoreductase) a unique multi-copper oxidase enzyme has been studied rigorously since its identification. However, there is ambivalence associated with various aspects of laccase, e.g., assay conditions and calculations. Our aim was to minimize its ambivalence, thus, total of five formulas (F1-F5) wereused to determine laccase activity of white and blue laccase. In case of enzymatic profiling of blue laccase, its activity ranged from 0.04 to 464.3UL-1 whereas in case of white laccase it ranged from 0.05 to 1404.7UL-1. The affinity of laccase at various enzyme concentration (0.3-0.9mgmL-1), time (5 and 10min) along with various substrates, i.e., 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol (GCL), 2,6-dimethoxyphenol (DMP) and syringaldazine (SYZ), and its concentration (ABTS 0.5-1.5mM, GCL 20-30mM, DMP 1-5mM, SYZ 10-30mM) were inferred. The optimal substrate concentrations were 1.5 and 0.5mM ABTS for blue and white laccase, respectively, with 30mM GCL and 2mM DMP being the common parameter. The optimal substrate concentrations were0.5mM ABTS, 20mM GCL, 1mM DMP and 30mM SYZ for commercial laccase. It was observed that the optimal protein load and reaction time was 0.3mgmL-1 and 5min in all the cases, however, in case of white laccase it was 0.6mgmL-1 at 10min for DMP and in case of commercial laccase it was 0.9mgmL-1 and 5min for SYZ. In the present study, F1 was most appropriate among the five formula used as it incorporates all the significant factors and use of single formula will help reduce future ambiguity.

Production of Recombinant Human Asparaginase from Escherichia coli under Optimized Fermentation Conditions: Effect of Physicochemical Properties on Enzyme Activity

The recombinant human asparaginase (rhASP) plays an important role in the treatment of acute lymphoblastic leukemia. In the present work, volumetric mass transfer coefficient (kLa) values are derived from the E. coli cultivation under different agitation and aeration conditions, in order to improve the rhASP productivity. The aeration and agitation conditions were systematically optimized by the kLa. The maximum biomass (2.4 g/L) and rhASP (1.68 g/L) are achieved with the kLa of 0.024 s-1 at 1.5 lpm and 700 rpm process conditions. The kinetic properties of purified rhASP are also extensively studied and optimized for the maximal enzyme activity. The optimal pH, temperature and incubation duration conditions for accomplishing maximum enzyme activity are found to be 9.0, 40°C, and 30 min, respectively. The optimum substrate concentration and substrate specificity for the highest enzyme activity are of 0.07 M and L-asparagine, respectively. The enzyme activity (204 IU/mL) is significantly improved in the presence of sodium metal (Na+) ions and the inhibitors 2- mercaptoethanol, bromoacetic acid and urea have presented the highest inhibition rate on rhASP activity. The enzyme kinetic parameters Km and Vmax of the purified rhASP are recorded as 2.25 mM and 250 IU/mL, respectively. This work provides the extensive characteristic properties of rhASP enzyme, which enables us to place in a competition for the development of oncology drugs.

Utilization of Crude Intracellular Chitinase Enzyme from Providencia stuartii for Glucosamine Production from Shrimp Shells

Chitin hydrolysis using enzyme is one of the methods to produce glucosamine in shorter time compared to using microbial cells, but the ability to produce glucosamine at enzyme’s optimum condition is influenced by substrate concentration and fermentation time. The objective of this research was to determine the optimum substrate concentration and fermentation time of shrimp shells’ chitin to produce glucosamine at the optimum pH and temperature of crude intracellular chitinase enzyme from Providencia stuartii. Method used was experimental method, started by extraction of intracellular enzyme from P. stuartii, followed by determination of optimum pH and temperature of enzyme. The optimum condition was used for experiment of shrimp shells’ chitin fermentation with treatments of chitin substrate concentration (0.5; 1.0; 1.5; 2.0%) and fermentation time (2, 4, 6 and 24 hours). Results showed that optimum enzyme activity occurred at pH of 5.0 and temperature of 40oC, which was about 6.03 U/ml. Concentration of chitin substrate and fermentation time influenced the amount of glucosamine obtained. Fermentation of shrimp shells’ chitin using crude intracellular enzyme was optimum at 1.0% substrate concentration and 6 hours fermentation time, which produced glucosamine about 1680.06±58.49 ppm. Keywords: intracellular chitinase enzyme, glucosamine, shrimp shells’ chitin, P. stuartii

Purification and Characterization of Metalloprotease from Serratia marcescens PPB-26 and Its Application for Detergent Additive

In this study, the extracellular metalloprotease from Serratia marcescens PPB-26 was purified to homogeneity via ethanol fractionation and DEAE-cellulose column chromatography. Thus, a 3.8-fold purification was achieved with a 20% yield and specific activity of 76.2 U/mg. The purified protease was a 50-kDa monomer whose optimum pH and temperature for activity were 7.5 and 30℃ respectively; however, it was found to remain active in the 5-9 pH range and up to 40℃ for 6 h. The protease had a half-life of 15 days at 4℃, an optimum reaction time of 10 min, and an optimum substrate (casein) concentration of 0.25%. Furthermore, the Michaelis constant (Km) and reaction velocity (Vmax) of the protease were calculated to be 0.28% and 111.11 μmoles/(min·mg)-1, respectively. The protease was stable when subjected to metal ions (2 mM), showing increased activity with most (especially CoCl2 and MgSO4 (30.54% increase)). It was also stable when exposed to oxidizing agents, bleaching agents, and detergents (5% v/v for 60 min). It retained 93% of its activity in non-ionic detergents (Tween-20, Tween-80, and Triton X-100). Moreover, wash performance analysis in commercial detergents (Ariel and Tide) showed that not only was the protease capable of protein stain removal, but also reduced cleaning time by 80% when added to detergents. Thus, the Serratia marcescens PPB-26 metalloprotease appears to be a promising new candidate as a laundry additive in the detergent industry.

Highly efficient asymmetric reduction of 2-octanone in biphasic system by immobilized Acetobacter sp. CCTCC M209061 cells

Highly efficient asymmetric reduction of 2-octanone to (R)-2-octanol catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells was achieved in a biphasic system. Bioreduction conducted in aqueous single phase buffer was limited due to poor solubility and toxicity towards cells cause by product accumulation. Introduction of [C4MIM]·Ac accelerated the biotransformation process, giving 99% yield, >99% product e.e. and 1.42-fold higher initial reaction rate in conversion of 10 mM 2-octanone as substrate, compared with 99% yield, 97.3% product e.e. and 1.57 μmol min−1 of initial reaction rate in a aqueous single phase buffer system containing 6 mM 2-octanone as the optimal substrate concentration. Moreover, in the [C4MIM]·Ac-containing buffer/n-tetradecane biphasic system, the optimal substrate concentration was enhanced by 83 times (500 mM) in comparison with that in aqueous single phase buffer, resulting in 53.4% yield (267 mM), 99% product e.e. with 8.9 mM g-1 h-1 space time yield.

Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

This study was done to partially purification of topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Biosynthesis of biologically active chitinase utilizing some Egyptian chitinaceous wastes and the properties of the synthesized enzyme

Background and objective Chitin-degrading enzymes have an utmost practical importance in many fields such as medicine, agriculture, and industry. These enzymes are used as effective antibacterial, antifungal, antihypertensive, and antioxidant agents and also as excellent food quality enhancers. The objective of the present article was to formulate the production medium and to pinpoint the proper growth conditions for the chosen microorganism producing highly active chitinase enzymes. The general properties of the crude enzyme preparation were determined to define the proper conditions for enzyme action. Under the specified conditions, the capability of the enzyme preparation for antimicrobial and antioxidant activities were decided. Materials and methods Eighteen recommended microbial strains were screened for biologically active chitinolytic enzymes productivity. Chitinase enzyme was determined, and also the important properties of the crude chitinase were duly pinpointed. Finally, biological activities of the crude enzyme were studied. Results and conclusion and conclusion Among all the 18 organisms, Streptomyces halstedii H2 was the most potent producer of an influential chitinase enzyme. The maximum chitinase activity of 49.5 U/ml was obtained from medium contains glucose 6 g/l, ammonium nitrate (0.9 g/l), and urea (0.64 g/l) at 30°C and pH 9.0. The important properties of the streptomycetal chitinase were duly pinpointed as follows: optimum enzyme and substrate concentrations were 1.6 mg/ml and 1.4% (w/v), respectively, and optimum reaction pH and temperature were 7.2 and 45°C, respectively. The crude preparation was stable for 60 min at pH 7.2 and 30°C and retained 92.6% of the original activity. Under the specified conditions, at varying concentrations, the enzyme preparation exhibited considerable 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity accompanied with low antimicrobial activity, pointing out the partial purification necessity of the crude enzyme preparation.

Co-Digestion of Napier Grass with Food Waste and Napier Silage with Food Waste for Methane Production

Enhancement of methane production by co-digestion of Napier grass and Napier silage with food waste was investigated in batch and repeated batch modes. First, the ratios of Napier grass to food waste and Napier silage to food waste were varied at different g-volatile solids (VS) to g-VS at an initial substrate concentration of 5 g-VS/L. The optimum ratios of Napier grass to food waste and Napier silage to food waste were 1:4 and 3:2 (g-VS/g-VS), respectively. This gave maximum methane yields (MY) of 411 and 362 mL-CH4/g-VSadded, respectively. Subsequently, the suitable ratios were used to produce methane at various substrate concentrations. A maximal MY of 403 and 353 mL CH4/g-VS were attained when concentrations of Napier grass co-digested with food waste and Napier silage co-digested with food waste were 15 g-VS/L and 20 g-VS/L, respectively. Under the optimum substrate concentration, the maximum MY from co-digestion of Napier grass with food waste was 1.14 times higher than that of Napier silage with food waste. Thus, co-digestion of Napier grass with food waste was further investigated at various organic loading rates (OLRs) in a 10.25 L horizontal reactor with a working volume of 5 L at an optimal ratio of 1:4 (g-VS/g-VS) and substrate concentration of 15 g VS/L. An OLR of 1.5 g-VS/L∙d gave a maximum methane production rate and MY of 0.5 L CH4/L∙d and 0.33 L-CH4/g-VSadded, respectively. Under the optimum OLR, the predominant methane producers were Methanoregula sp., Methanotorris sp., Methanobacterium sp., Methanogenium sp. and Methanosarcina sp. An energy production of 11.9 kJ/g-VSadded was attained.

Highly Efficient Synthesis of 2,5-Dihydroxypyridine using Pseudomonas sp. ZZ-5 Nicotine Hydroxylase Immobilized on Immobead 150

In this report, the use of immobilized nicotine hydroxylase from Pseudomonas sp. ZZ-5 (HSPHZZ) for the production of 2,5-dihydroxypyridine (2,5-DHP) from 6-hydroxy-3-succinoylpyridine (HSP) in the presence of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) is described. HSPHZZ was covalently immobilized on Immobead 150 (ImmHSPHZZ). ImmHSPHZZ (obtained with 5–30 mg of protein per gram of support) catalyzed the hydrolysis of HSP to 2,5-DHP. At a protein loading of 15 mg g−1, ImmHSPHZZ converted 93.6% of HSP to 2,5-DHP in 6 h. The activity of ImmHSPHZZ was compared with that of free HSPHZZ under various conditions, including pH, temperature, enzyme concentration, substrate concentration and stability over time, and kinetic parameters were measured. The results showed that ImmHSPHZZ performed better over wider ranges of pH and temperature when compared with that of HSPHZZ. The optimal concentrations of ImmHSPHZZ and substrate were 30 mg L−1 and 0.75 mM, respectively. Under optimal conditions, 94.5 mg L−1 of 2,5-DHP was produced after 30 min with 85.4% conversion. After 8 reaction cycles and 6 days of storage, 51.3% and 75.0% of the initial enzyme activity remained, respectively. The results provide a framework for development of commercially suitable immobilized enzymes that produce 2,5-DHP.

Characterization of cross-linked amyloglucosidase aggregates from Aspergillus fumigatus KIBGE-IB33 for continuous production of glucose

Current research deals with immobilization of amyloglucosidase through carrier-free approach using cross-linking strategy. Cross-linked amyloglucosidase aggregates (CLAAs) with aggregation yield of 94% were prepared in 04 h by incorporating 40% ammonium sulfate and 1.5% glutaraldehyde in enzyme solution. CLAAs were characterized by optimizing various conditions including reaction time, pH, temperature and substrate concentration. It was noticed that after cross-linking no change in optimum reaction time and substrate concentration was observed however, a 5-degree shift in optimum temperature from 60 °C to 65 °C was obtained as compared to soluble amyloglucosidase. Activation energy (Ea) of amyloglucosidase as calculated from Arrhenius plot was 5.5 kcal mol−1 and 5.2 kcal mol−1 for soluble and cross-linked aggregates, respectively. Stability studies revealed that CLAAs can be used at higher temperatures for longer time period than soluble amyloglucosidase. Furthermore, data of recycling studies showed that CLAAs can be efficiently reused for 20 cycles with the retention of 63% of its initial activity. Due to the continuous reusability of CLAAs, the product formation is also increased 8 times from 5.71 mg ml−1 (soluble enzyme) to 46.548 mg ml−1 (CLAAs). Findings of this research show that carrier-free strategy is more effective for continuous hydrolysis of starch and production of glucose.