Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

Purification and characterization of a mutant ATP phosphoribosyltransferase hypersensitive to histidine feedback inhibition.

A mutant form of ATP phosphoribosyltranferase (EC 2.4.2.17), hisG1708c, which results in abnormally slow growth of Salmonella typhimurium at 20 °C was purified to homogeneity and kinetic and chemical behavior were characterized. Initial velocity steady-state substrate kinetics of wild-type and mutant enzymes at 37 °C were consistent with sequential kinetics and demonstrated that standard assay concentrations of substrates were sufficient to substantially saturate both enzymes. Nearly time-independent inhibition by histidine at 37 °C could be obtained only after incubation in the presence of product and histidine. Studies at 37 °C showed that the mutant enzyme is 24 times more sensitive to histidine than the wild type in a negatively cooperative manner instead of the positively cooperative manner seen for wild type. Pure mutant enzyme exhibits two major electrophoretic species of native enzyme. Although one less cysteine is titratable in native mutant enzyme, the amino acid compositions of mutant and wild-type enzymes are similar. Histidine produces an ultraviolet difference spectrum in mutant enzyme closely resembling that produced in wild type. Binding of histidyl-tRNA to mutant enzyme is substantially inhibited by histidine. It is concluded that the hisG1708c mutation alters some conformational processes coupled to the histidine binding site while not affecting others.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Development of mutants of <Emphasis Type="Italic">Melanocarpus albomyces</Emphasis> for hyperproduction of xylanase

The wild type filamentous fungus, Melanocarpus albomyces, produces many commercially valuable enzymes, including Xylanases and Xylan-debranching enzymes with low activity. In this paper, we report for the first time the development of M. albomyces mutants from vegetative spores. Profuse sporulation of M. albomyces was induced on Potato Carrot Agar medium. These spores, when subjected to chemical mutation, led to the isolation of the hyper-xylanase producing mutant, viz, M. albomyces IITD3A. Various parameters including number of spores, nitrogen source and C/N ratio of the medium were optimized for production of xylanase by the mutant in a shake flask culture. Under controlled pH at 7.8, the mutant produced highly active xylanase with 415 IU/mL after 36 h of growth on soluble alkaline lignocellulosic extract in a 14-L fermentor. The overall productivity of xylanase was 8-fold higher than the wild type culture with11, 530 IU/L/h. The enzyme can be easily stored at 37°C for 50 days by addition of a small amount of the preservative — thiomersal. Also, for long term storage, a lyophilized powder form of the enzyme can be used which retained 100% of its activity for > 50 days. When assayed at pH 7.5 and temperature 55°C, the xylanase retained 100% of its original activity, and also at pH 9.0, it retained > 50% of its activity for 2 h, which is promising for its application in the pulp and paper industry.     

Temperature effects on endogenous indole-3-acetic acid levels in leaves and stamens of the normal and male sterile 'stamenless-2' mutant of tomato (Lycopersicon esculentum Mill.)

The indole-3-acetic acid (IAA) concentration in leaves and stamens of the normal and a temperature-sensitive male sterile ‘stamenless-2′ (sl-2/sl-2) mutant of tomato (Lycopersicon esculentum Mill.), grown under three temperature conditions, was measured by gas chromatography — mass spectrometry — selected ion monitoring (GC-MS-SIM) and by enzyme-linked immunosorbant assay (ELISA). At low (LTR, 18°C day/15°C night), intermediate (ITR, 23°C day/18°C night), and high temperatures (HTR, 28°C day/23°C night), the mutant leaves had approximately 10 to 20 times higher IAA concentrations, respectively, than the normal leaves under these temperature regimes. Similarly, the stamens of mutant flowers had approximately five and eight times higher IAA concentration at ITR and HTR, respectively, than the normal flowers. In the low temperature reverted mutant stamens, however, the level of IAA was similar to that in normal stamens. Also, with an increase in temperature, there was an increase in the level of IAA in the leaves and stamens of mutant plants. However, different temperatures had no appreciable effect on the IAA content of leaves and stamens of normal plants. It is suggested that the high IAA content in leaves and stamens of the stamenless-2 mutant is one of the factors associated with male sterility and carpellization of stamens in this mutant.     

Biochemical and Structural Characterization of Two Site-Directed Mutants of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42°C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80°C all enzymes retained their initial activities.     

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t _1/2) inactivation at 65 °C, 18 °C increase in apparent T _m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

     

Directed Mutagenesis of the Conserved Asparagine Residues of Bacillus Stearothermophilus Leucine Aminopeptidase II

Summary To understand the structure–function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of the four conserved asparagine residues was replaced with leucine, aspartate, and lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular mass of approximately 44.5 kDa were purified to homogeneity by nickel-chelate chromatography. Substitution of Asn-245, Asn-335, and Asn-341 with Lys generated variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanilide as the substrate revealed an increase in k_cat with no significant change in K_m, leading to a more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335, Asn-341, and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70 °C, indicating that the deamination of these residues may have a deleterious effect on LAPII.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

Stability of the Cellulase of Trichoderma reesei under use conditions

Enzyme stability studies have been reinvestigated under the conditions used for cellulose hydrolysis (pH 4.8, 50°C, 24 hr). The cellobiohydrolase (CBH) component as measured on Avicel is less stable than other enzymes of the cellulase complex, and is 60% inactivated by merthiolate (and other Hg compounds) under the above conditions. Endo-β-1,4-glucanase is much more stable, and more resistant to merthiolate and other compounds. Under unshaken conditions the Avicelase of the Rutgers strain C 30 shows greater stability to heat than that of other available strains. Biocides must be selected not only for their ability to prevent contamination, but also for their compatibility with cellulases. Tetracycline and chlortetracycline are inexpensive, effective in very low concentrations, have no harmful effect on the enzymes, and are compatible with the yeasts that subsequently grow on the sugar solutions to produce alcohol. Attempts have been made to stabilize the enzymes by chemical modification in such a way as to maintain their solubility. Glutaraldehyde treatment greatly increased the enzyme size, lowered the pI values, and gave a slight shift in the pH activity curve. There was, unfortunately, no increase in enzyme stability, and the activity of enzymes on solid celluloses was adversely affected. Shaking greatly reduced the hydrolysis of Avicel by Trichoderma reesei C 30 enzyme. The adverse effect was accompanied by a decrease in recoverable enzyme and protein.     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens α-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K _m value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K _cat/K _m) and thermo-stability at 60°C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10°C and 3–4 units, respectively.     

Production of highly active fungal milk-clotting enzyme by solid-state fermentation

Abstract

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383?±?486?U/ml and 16,373?± 558?U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30?g wheat bran containing 6% defatted soy meal at 30?°C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5–10 and temperature 45–55?°C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30?g wheat bran containing 4% defatted soy meal at 30?°C, pH 6) and the enzyme was active from pH 6–9 at 50?°C. Curd yields prepared from fungal enzyme extract decreased (5–9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet     

Introduction of a disulfide bridge enhances the thermostability of a <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> xylanase mutant

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.     

Enhanced thermostability of keratinase by computational design and empirical mutation

Keratinases are proteolytic enzymes capable of degrading insoluble keratins. The importance of these enzymes is being increasingly recognized in fields as diverse as animal feed production, textile processing, detergent formulation, leather manufacture, and medicine. To enhance the thermostability of Bacillus licheniformis BBE11-1 keratinase, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Use of the algorithm in combination with molecular modification of homologous subtilisin allowed the introduction of four amino acid substitutions (N122Y, N217S, A193P, N160C) into the enzyme by site-directed mutagenesis, and the mutant genes were expressed in Bacillus subtilis WB600. The quadruple mutant displayed synergistic or additive effects with an 8.6-fold increase in the t _1/2 value at 60 °C. The N122Y substitution also led to an approximately 5.6-fold increase in catalytic efficiency compared to that of the wild-type keratinase. These results provide further insight into the thermostability of keratinase and suggest further potential industrial applications.     

Comparison of Two C-P Bond-forming Enzymes Involved in the Biosynthesis of Bialaphos

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158–163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 °C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 °C of HPH to 53 and 58.8 °C respectively. Specific activities and steady-state kinetics measured at 25 °C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.     

Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum

A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability. In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature of XynX-C was about 5–10 °C lower than that of XynX-TC. Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Biochemical characterization and evaluation of invertases produced from Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa for the production of fructooligosaccharides

Abstract

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70?°C for Rhodotorula mucilaginosa invertase and 4.5 and 50?°C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75?°C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50?°C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3?g/L of nystose by S. cerevisiae invertase and 12.6?g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.     

Development of efficient strain of Ganoderma lucidum for biological stripping of cotton fabric dyed Reactive Blue 21

One of the most common dyeing problems of textile industries is uneven and faulty dyeing over the finished quality of fabrics due to different reasons. These problems are usually tackled through chemical degradation in which uneven and faulty dye is removed from the surface of fiber but fabric quality is compromised. Chemical process also reduces the strength of the fabric and durability of textile material by reduction in reactive dye ability. The fabric cannot be reused due to the reduced strength. To overcome above mentioned problem, biological method of stripping in which enzymes produced by different micro-organisms are used. This process has no harmful effect on the fabric and is safe for environment. In this research work reactive blue 21 dye with 0.5, 2 and 4% shade strengths was used to dye cotton fabric. The Ganoderma lucidum fungal strains were mutated by UV mutagen, and five were selected for further processing. These mutant strains were grown at temperature ranges (20 °C to 40 °C); pH(3–5); inoculum size(1–5 mL) and fermentation time (3–15 days) . The required nutrients media to produce the ligninolytic enzymes was added to the flask. The strain which gave the fast decolourization results was selected for further optimization. Optimization was done by observing the variables: incubation time 12 days, pH 4, temperature 30 °C, and inoculum size 3 mL by applying Response Surface Methodology (RSM) in Central Composite Design (CCD). During the process of fabric color stripping, the enzyme assay revealed that the respective mutant UV-60 strain produced active enzymes with their V_max, Mnp (427U/mL), LiP (785U/mL), and Lac (75 U/mL) enzymes decolorized 89% of the dye which is 25% more than the parent strain and also the production of enzyme is Mnp (344U/mL), LiP (693U/mL), and Lac (59 U/mL) enzymes which is lower than mutant strain.