Glucose Isomerase Production Research Articles

Coculture and Immobilization of Cellulolytic Bacteria for Enhanced Glucose Isomerase Production from Wheat Straw

Coculture and Immobilization of Cellulolytic Bacteria for Enhanced Glucose Isomerase Production from Wheat Straw

Isolation and Screening of Aspergillus niger and Bacillus coagulans as Potential Candidates for Amylase and Glucose Isomerase Production

There is an increasing demand for high fructose corn syrup as an alternative to glucose especially for use by people with diabetes due to its low glycemic index. The high cost of this product coupled with its high demand has attracted the attention of many researchers to search for an alternative and sustainable production route. Therefore, the focus of this work was isolating and screening Aspergillus niger and Bacillus coagulans as possible producers of amylase and glucose isomerase respectively, which are needed to produce high fructose corn syrup. Nine (9) samples of loamy soil were taken from three (3) separate locations (BG = Botanical Garden, RD = Refuse Dumpsite, FB = Flower Bed) to isolate the bacterium and fungus. Based on the lacto-phenol cotton blue stain preparation under a microscope, colonies that were thought to be Aspergillus niger colonies were observed and further identified. Cultural, microscopic, and biochemical characteristics were used to confirm the identification of colonies that were thought to be Bacillus coagulans. The probable isolates of Aspergillus niger and Bacillus coagulans were then screened for amylase and glucose isomerase production respectively. Out of the total of nine (9) soil samples analysed, 5 (55.6 %) were positive for Aspergillus niger. A higher isolation rate of Aspergillus niger, 2 (66.7 %) was recorded in soil samples from botanical garden (BG) and flower bed (FB). The lowest isolation rate, 1 (33.3 %) was observed in soil obtained from the refuse dump. Out of all the isolates screened for their potential to produce amylase, the isolate from the botanical garden showed the highest zone of starch hydrolysis (28 mm), and the isolate from the flower bed showed the least zone of hydrolysis of starch (13 mm). On the other hand, out of the nine (9) soil samples analysed, 6 (66.7 %) were positive for Bacillus coagulans and the highest occurrence of Bacillus coagulans, 3 (100 %) was recorded with soil from botanical garden (BG1), while the least occurrence, 1 (33.3 %) was observed in soil from flower bed (FB2). The probable isolates of Bacillus coagulans screened for glucose isomerase production revealed that all the six (6) isolates produced glucose isomerase, with isolate from refuse dump (RD2) producing the highest concentration of glucose isomerase (4.7014 g/L).

Production of Glucose Isomerase from Streptomyces roseiscleroticus

This study was aimed at the isolation and characterization of a microbial strain capable of producing glucose isomerase. Microbial strain was isolated from soil using starch casein agar as a differential media. The isolated microbial strain was capable of producing glucose isomerase which was tested using 2, 3, 5 - triphenyltetrazolium solution as a chromogenic substrate. The microbial strain was identified as Streptomyces species based on its morphological and microscopic characteristics. It was further subjected to molecular characterization using 16S rRNA sequencing and was subsequently confirmed as Streptomyces roseiscleroticus. Glucose isomerase was produced from Streptomyces roseiscleroticus after 120 hr of submerged fermentation at pH 6.8 and at 37°C utilizing xylose as the sole carbon source and a compendium of peptone, beef and yeast extracts as nitrogen sources. These findings suggest that the microbial strain, Streptomyces roseiscleroticus can be a useful bacterial source for the production of glucose isomerase needed for commercial and industrial utilization.

Production and application of glucose isomerase from Streptomyces enissocaesilis and amylase from Streptomyces sp. for the synthesis of high fructose corn syrup

This study reports Streptomyces sp. strain T.S.A.KP identified as Streptomyces enissocaesilis isolated from soil inhabiting actinobacteria of Madhya Pradesh (India) as a new actinobacterial strain for production of glucose isomerase (GI) and Streptomyces sp. strain TSA. Dm1 closely related to Streptomyces sampsonii for the production of amylase. These strains were selected from an array of 120 actinobacterial isolates, which on screening originally yielded 18% GI producers and 36% amylase producers. S. enissocaesilis yielded maximum GI at pH 7 and 70 °C in 120 h. Highest amylase production from Streptomyces sp. strain TSA. Dm1 was observed at pH 7 and 37 °C in 96 h. Michaelis–Menten constant (Km) for GI and amylase was 1.54 M and 0.36 M, respectively. Vmax for GI was 11.76 U/ml and amylase 33 U/ml. These cultures were employed for HFCS production. Saccharification of corn starch was achieved up to 90.9% by Streptomyces sp. strain TSA. Dm1, and the glucose was isomerized to 40.8% by S. enissocaesilis. Presence of glucose and fructose in HFCS was also confirmed by high-performance liquid chromatography.

Optimization of various encapsulation systems for efficient immobilization of actinobacterial glucose isomerase

Abstract Glucose isomerase (GI) converts glucose into fructose by a reversible reaction. This reaction is industrially valuable for the production of high fructose corn syrup (HFCS) which is used as a sweetener by food and beverage industries. GI being an expensive enzyme necessitates its usage in immobilized form for increasing its reusability and economic viability. Global HFCS production accounts to 10 million tons which is highest among products yielded by immobilized process. Current study evaluates different encapsulation systems for immobilization of GI produced by Streptomyces sp. T.S.A.KP which was found to be Streptomyces enissocaesilis (Accession number MN911386 ). This is the first report of extracellular GI production by a strain of Streptomyces enissocaesilis. GI was immobilized using chitosan, alginate and agar to enhance its stability and conversion efficiency. The concentration of polymeric gel with cross linking agent was optimized to get ideal pore size for maximum GI activity. Immobilization on chitosan significantly increased (P

Purification and kinetic behavior of glucose isomerase from Streptomyces lividans RSU26.

Glucose isomerase (GI), an enzyme with deserved high potential in the world market. GI plays a major role in high Fructose Corn Syrup Production (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of various industrially valuable enzymes, including Glucose isomerase. Currently, recombinant strains have been available for the production of various enzymes, but it has limitation in the large scale production. Therefore, identifying effective streptomyces strains have emerged. The current study, the novel S. lividans RSU26 was isolated from a marine source and optimized its potential to produce glucose isomerase at different physical and chemical conditions. The optimum pH and temperature for GI and biomass production were 7.5 and 35 °C, respectively at 96 h. Characterization study revealed that the approximate molar mass of GI was 43 kDa for monomeric and 170 kDa for tetrameric forms. Kinetic behavior exhibits Km, and Vmax values for the conversion of fructose to glucose conversion were 48.8 mM and 2.54 U mg−1 at 50 °C and glucose to fructose were 29.4 mM and 2.38 U mg−1 at 65 °C protein, respectively. Therefore, the present study suggested that the wild–type S. lividans RSU26 has strong potential to produce glucose isomerase for various industrial applications.

Marker‐Free System Using Ribosomal Promoters Enhanced Xylose/Glucose Isomerase Production in Streptomyces rubiginosus

Xylose/glucose isomerases are important industrial enzymes that are most widely used in food industries; however, their previously reported expression levels do not meet the requirements for industrial application. Here, an antibiotic resistance marker (ARM)-free system driven by ribosomal RNA (rRNA) promoters is developed to obtain high-level xylose/glucose isomerase (XI/GI) expression in Streptomyces rubiginosus (S. rubiginosus). The rRNA promoter rrnD yields the highest glucose isomerase production titer of XIs/GIs, which is eight times higher than that of ermEp* and 2.6 times higher than that of kasOp*. The integrated ARM gene is removed by further introduction of the Cre plasmid with a temperature-sensitive replicon. The production titer of XIs/GIs is further improved by replacing the xylR gene with an additional expression glucose isomerase cassette at the xylR locus. Ultimately, the glucose isomerase activity reaches up to 79.7 ± 7.5 U mL-1 at 96 h. The results support the robustness and stability of XI/GI production with this ARM-free system using optimal ribosomal promoters in S. rubiginosus, demonstrating strong potential in large-scale industrial applications. Besides, the results imply that rRNA promoters are strong promoters that can be used for protein engineering or metabolic engineering.

Screening of Xylanase and Glucose Isomerase Producing Bacteria Isolated from Hot Springs in Turkey

The aim of this study was screening of xylanase and glucose isomerase producing thermophilic bacteria isolated from some hot springs located to Aegean Region of Turkey. Total sixty eight thermophilic isolates (Anoxybacillus, Brevibacillus, Geobacillus, Aneurinibacillus, Thermus, Paenibacillus and Proteobacter) were collected previously from these fields and identified based on 16S rDNA gene sequences. The isolates were screened with plate assay for determining the xylanase and glucose isomerase production abilities seperately in order to find new strains for industrial processes. After an incubation period of two days for xylanase and 5-6 days for glucose isomerase at 50-60 °C, positive strains were determined. Enzyme producing strains were confirmed by spectrophotemetric measurements with crude enzyme extracts, birchwood xylan and glucose were used as substrates. Most of the strains (59 strains) were positive for xylan degradation while only sixteen of the strains had showed glucose isomerase activity. Fourteen of the strains have showed both xylanase and glucose isomerase activity. None of the Paenibacillus, Aneurinibacillus, and Proteobacter strains were glucose isomerase positive, although the glucose isomerase activity of Geobacillus strains were notably high. Both xylanase and glucose isomerase activities have observed at 50-60 °C which is suitable for biotechnological applications.

Production, Characterization and Optimal Performance Studies of Glucose Isomerase by Achromobacter xylosoxidans mck-4 Isolated from Starch Milling Wastes

Production, Characterization and Optimal Performance Studies of Glucose Isomerase by Achromobacter xylosoxidans mck-4 Isolated from Starch Milling Wastes

Optimization of Fermentation Conditions and Media for Production of Glucose Isomerase from Bacillus megaterium Using Response Surface Methodology.

Glucose isomerase is an enzyme widely used in food industry for producing high-fructose corn syrup. Many microbes, including Bacillus megaterium, have been found to be able to produce glucose isomerase. However, the number of studies of glucose isomerase production from Bacillus megaterium is limited. In this study, we establish the optimal medium components and culture conditions for Bacillus megaterium glucose isomerase production by evaluating the combined influence of multiple factors and different parameters via Plackett–Burman design and response surface methodology in Modde 5.0 software. The optimized conditions, which were experimentally confirmed as follows: D-xylose (1.116%), K2HPO4 (0.2%), MgSO4·7H2O (0.1%), yeast extract (1.161%), peptone (1%), pH 7.0, inoculum size 20% (w/v), shaking 120 rpm at 36.528°C for 48 hours, give rise to production of highest activity of glucose isomerase (0.274 ± 0.003 U/mg biomass). These results provide additional important information for future development of large-scale glucose isomerase production by Bacillus megaterium.

Glucose Isomerase Production and Its Application InVarious Fields

Glucose Isomerase Production and Its Application InVarious Fields

Beet molasses-based feeding strategy enhances recombinant thermostable glucose isomerase production by Escherichia coli BL21 (DE3).

The aim of this work was to develop an effective fed-batch feeding strategy to enhance recombinant glucose isomerase (r-GI) production by recombinant Escherichia coli BL21 (DE3) pLysS on an industrially relevant feedstock without the application of an exogenous inducer. Following the batch operation (0 < t < 7H), the effects of pulse and/or continuous feeding of hydrolyzed beet molasses were investigated under five different feeding strategies. The two most promising strategies with respect to r-GI activity were (i) PM-0.05, designed with one pulse feed (t = 7H) followed by a continuous feed and (ii) 2PMF -0.05, designed with two consecutive pulse feeds (t = 7 and 10H) followed by a continuous feed. The continuous feeding of molasses for both fermentation strategies employed the same precalculated feeding rate, μo = 0.05H-1 . The maximum r-GI activities exhibited by PM-0.05 and 2PMF -0.05 were 29,050 and 30,642U dm-3 , respectively. On the one hand, compared to PM-0.05 r-GI activity reached its maximum within a shorter cultivation time (∆tmax = 2H) at 2PMF -0.05, which could be preferable in terms of manufacturing costs and possible risks; on the other hand, PM-0.05 is a simpler fermentation regime compared to 2PMF -0.05 with respect to manipulations that should be considered in large-scale production.

Studies on the production of glucose isomerase by Bacillus licheniformis

Abstract This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein). Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively). The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein). Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein). In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein) while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein). The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

Codon optimization of xylA gene for recombinant glucose isomerase production in Pichia pastoris and fed-batch feeding strategies to fine-tune bioreactor performance.

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30% of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5gL(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5gL(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117gL(-1) at t=66h and 32530UL(-1) at t=53h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t=53h as 0.21gg(-1) in S5 strategy.

Recombinant protein production by sucrose‐utilizing Escherichia coli W: untreated beet molasses‐based feeding strategy development

AbstractBACKGROUNDUntreated beet molasses feeding strategy was developed for recombinant thermostable glucose isomerase (GI) production by sucrose‐utilizing Escherichia coli W.RESULTSpRSETA::xylA plasmid carrying GI encoding gene of Thermus thermophilus (xylA) was transferred into E. coli W, and the effects of carbon sources, i.e. glucose, sucrose, and beet molasses containing 50% sucrose, on GI production were investigated. 32 g L−1 beet molasses‐based medium resulted in the highest recombinant GI production (A = 4364.1 U L−1) and cell concentration (CX = 4.2 g L−1) in laboratory‐scale shake‐bioreactors. Using beet molasses as the carbon source, pulse and exponential feeding strategies were designed. The highest recombinant GI production was achieved as 35 265 U L−1 at t = 16 h in V = 3.0 L controlled bioreactors, where the cell concentration was CX = 17 g L−1, by the application of a three‐stage strategy: Stage I (0&lt;t&lt;5 h): batch operation with the beet molasses and (NH4)2HPO4 initial concentrations of CM0 = 32 g L−1 and CN0,(NH4)2HPO4 = 5 g L−1, respectively; Stage II (5≤t&lt;11 h): semi‐batch operation by consecutive pulse feeding of (NH4)2HPO4 containing molasses at t = 5 h and t = 8 h, to increase CM to 32 g L−1 and C (NH4)2HPO4 to 5 g L−1; and Stage III (t ≥11 h): semi‐batch operation by continuous feeding of molasses with the pre‐determined specific growth rate of μ0 = 0.05 h−1, starting at t = 11 h.CONCLUSIONSThis work demonstrates the significance of the use of beet molasses containing high concentrations of sucrose by designing semi‐batch feeding strategies for the over‐production of recombinant proteins by sucrose‐utilizing E. coli W. © 2014 Society of Chemical Industry

Production and partial characterization of extracellular glucose isomerase using thermophilic Bacillus sp. isolated from agricultural land

Abstract In this investigation, glucose isomerase was extracted from Bacillus sp., isolated from agricultural soil sample and purified further by ammonium sulfate fractionation followed by gel filtration chromatography using Sephadex G-25. The purified enzyme over crude was further subjected to partial characterization which includes the assessment of influence of pH, temperature, incubation time and metal ions on the stability and activity of glucose isomerase was observed. As a result, it was found that, glucose isomerase isolated from the thermophilic Bacillus sp. enumerated from the agricultural soil showed maximal stability at pH 8.0 and at temperature 70 °C within 35 min. It was also found that, at the minimum concentration of 5 mM Mg 2+ , Co 2+ and Mn 2+ showed 41%–59% of enzyme activity.

Glucose(xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues

Streptomyces sp. CH7 was found to efficiently produce glucose(xylose) isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose) isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its K m values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its V max values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85°C and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60°C after 30 min. These findings indicate that glucose(xylose) isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

AbstractBACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated.RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xylA) was recombined with pRSETA vector, and the pRSETA::xylA obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L−1 (20.6 U mg−1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h−1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h−1 operation as 9.6 g L−1 at t = 32 h. The highest oxygen uptake was 4.57 mol m−3 s−1 at M‐0.1 operation.CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry

Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1.

The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI), an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production.

Optimization of glucose isomerase production by Streptomyces albaduncus

The effect of environmental factors on glucose isomerase productivity of Saudi Arabia isolated Streptomyces albaduncus was studied. During growth of the tested microorganism in the basal medium at 30°C, the glucose isomerase production reached the maximum level (6530 U/L) after 96 h incubation period. Irrespective of the carbon source, replacement of xylose with different carbohydrate did not increase the enzyme productivity. On the other hand, the highest enzyme productivity was obtained when corn steep liquor (2.0%, w/v) and yeast extract (1%) were used as nitrogen sources. Also, the optimum initial pH for maximum growth and enzyme productivity was 7. Further improvement in the glucose isomerase production was obtained after the addition of 0.01% (w/v) DL-isoleucine to the culture medium. Accordingly, optimization of the environmental condition associated with increase in the glucose isomerase enzyme productivity from S. albaduncus up to 15328 U/L which is approximately 2.3 more times as compared to that obtained under the initial condition. Finally, a promising production of glucose isomerase (14100 U/L) at large scale level was achieved by cultivation of the tested strain in the optimum environmental conditions using laboratory fermentor of 14 L. Key words: Glucose isomerase, fermentation, environmental condition, Streptomyces albaduncus.