Partial purification and characterization of a new intracellular beta-glucosidase of Trichoderma reesei.
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ABSTRACT:
SUBMITTER: Inglin M
PROVIDER: S-EPMC1161380 | biostudies-other | 1980 Feb
REPOSITORIES: biostudies-other
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Project description:Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5-9.0 and possessed high thermal stability up to 50 °C with 80% of the residual activities compared with the stability seen at the pH range of 6.5-7.0 and temperatures of up to 40 °C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases.
| S-EPMC4720395 | biostudies-literature
Project description:?-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this ?-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. ?-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other ?-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus ?-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of ?-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred ?g/mL ?-D-glucose inhibited ?-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.
| S-EPMC6332095 | biostudies-literature
Project description:Background:The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as ?-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood. Results:Deletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular ?-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei. Conclusions:BGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.
| S-EPMC6240962 | biostudies-literature
Project description:The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.
| S-EPMC8123685 | biostudies-literature
Project description:BackgroundTrichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH.ResultsWe successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain.ConclusionsThe recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for β-glucosidase production as well as improving the biomass conversion using cellulases.
| S-EPMC3434039 | biostudies-literature
Project description:The fungus Trichoderma reesei grows on barley (Hordeum valgare L.) beta-glucan and pachyman, secreting increased quantities of exo-beta 1,3-glucanase. This enzyme is also found in commercial cellulase preparations, from which it has been partially purified. It has a mol.wt. of 700000, an isoelectric point of 4.2, is cold-stable and hydrolyses both beta 1 Leads to 3- and beta 1 Leads to 6-linkages.
| S-EPMC1162288 | biostudies-other
Project description:Backgroundβ-glucosidase is an important biomass-degrading enzyme and plays a vital role in generating renewable biofuels through enzymatic saccharification. In this study, we analyzed the transcriptome of Trichoderma harzianum HTASA derived from Hainan mangrove and identified a new gene encoding β-glucosidase Bgl3HB. And the biochemically characterization of β-glucosidase activity was performed.ResultsBgl3HB showed substantial catalytic activity in the pH range of 3.0-5.0 and at temperatures of 40 ℃-60 ℃. The enzyme was found quite stable at 50 ℃ with a loss of only 33.4% relative activity after 240 min of heat exposure. In addition, all tested metal ions were found to promote the enzyme activity. The β-glucosidase activity of Bgl3HB was enhanced by 2.12-fold of its original activity in the presence of 5 M NaCl. Surprisingly, Bgl3HB also showed a remarkable ability to hydrolyze laminarin compared to other measured substrates. Enzyme efficiency was examined in the sugarcane bagasse saccharification processes, in which Bgl3HB with 5 M NaCl worked better supplementing Celluclast 1.5L than the commercial Novozyme 188 ascertained it as an admirably suited biocatalyst for the utilization of agricultural waste. In this work, this is the first report of a halophilic β-glucosidase from Trichoderma harzianum, and represents the β-glucosidase with the highest known NaCl activation concentration. And adding 5 M NaCl could enhance saccharification performance even better than commercial cellulase.ConclusionsThese results show that Bgl3HB has great promise as a highly stable and highly efficient cellulase with important future applications in the industrial production of biofuels.
| S-EPMC9331182 | biostudies-literature
Project description:Sugar transporters play an important role in the cellulase production of lignocellulose-degrading fungi. Nevertheless, the role and function of these transporters are still unclear. Here we first report intracellular sugar transporters assisting cellulase production in Trichoderma reesei (T. reesei) using lactose. The mRNA levels of sugar transporter genes mfs, gst, and lac1 were substantially upregulated in T. reesei cultivated on lactose, with the most abundant mRNA levels at 24 h as compared to glucose. Moreover, the individual deletion of these sugar transporters significantly inhibited cellulase production, solid cell growth, and sporulation of T. reesei, suggesting they play a supporting role in cellulase production when grown in lactose. Surprisingly, MFS, GST, and LAC1 were mainly localized in the cytoplasm, with MFS and LAC1 in the endoplasmic reticulum (ER), representing the first discovery of intracellular sugar transporters involved in cellulase biosynthesis in lactose culture. The absence of the gene lac1 noticeably inhibited most of the crucial genes related to cellulase production, including cellulase-encoding genes, transcription factors, and sugar transporters, at 24 h, which was fully relieved at 48 h or 72 h, indicating that lac1 affects cellulase production more at the early step. This research advances the understanding of the function of intracellular sugar transporters in fungi, particularly for fungal cellulase production.
| S-EPMC9953249 | biostudies-literature
Project description:An extracellular beta-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5 A, alpha = 109.1, beta = 97.3, gamma = 114.5 degrees . The crystals diffracted to 1.8 A resolution using a rotating-anode generator and to 1.2 A resolution using a synchrotron source. On the basis of the Matthews coefficient (V(M) = 3.16 A(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme.
| S-EPMC2720328 | biostudies-literature
Project description:A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.
| S-EPMC1152451 | biostudies-other