Recent Advances in ß-Glucosidase Sequence and Structure Engineering: A Brief Review.

ß-glucosidases (BGLs) play a crucial role in the degradation of lignocellulosic biomass as well as in industrial applications such as pharmaceuticals, foods, and flavors. However, the application of BGLs has been largely hindered by issues such as low enzyme activity, product inhibition, low stability, etc. Many approaches have been developed to engineer BGLs to improve these enzymatic characteristics to facilitate industrial production. In this article, we review the recent advances in BGL engineering in the field, including the efforts from our laboratory. We summarize and discuss the BGL engineering studies according to the targeted functions as well as the specific strategies used for BGL engineering.

Enhancing cellulosic ethanol production through coevolution of multiple enzymatic characteristics of ß-glucosidase from Penicillium oxalicum 16.

In previous studies, we isolated a novel ß-glucosidase from Penicillium oxalicum 16 (16BGL), which is useful for producing cellulosic ethanol. However, 16BGL has a relatively low enzyme activity and product tolerance; besides, a huge gap exists between the optimum temperature of 16BGL (70 °C) and the fermentation temperature for producing cellulosic ethanol (40 °C). Here, we present a directed evolution-based study, which combines one-round error-prone PCR with three rounds of high-throughput screening, for coevolving multiple enzymatic characteristics of 16BGL. We identified an improved variant Y-1-B1 with a triple mutant (G414S/D421V/T441S). Y-1-B1 had an optimum temperature of 50 °C, much closer to the fermentation temperature. The catalytic efficiency of Y-1-B1 for hydrolyzing pNPG was 1355 mM-1 s-1 at 50 °C and pH 5, significantly higher than that of 16BGL (807 mM-1 s-1). Y-1-B1 also achieved a slightly reduced strength of product inhibition of 1.1 at a glucose concentration of 20 mM, compared with the ratio of 1.3 for 16BGL. A maximum titer of 6.9 g/L for ethanol production was achieved in the reaction with Y-1-B1, which was 22% higher than that achieved with 16BGL. Structure modeling revealed that the mutations are distant from the active-site pocket. Therefore, we performed molecular dynamics (MD) simulations to understand why these mutations can improve catalytic efficiency. MD simulation revealed that the nucleophilic residue Asp261 had a much closer contact with the glucosidic center of pNPG in the simulation with Y-1-B1 than that with 16BGL, suggesting that the mutant is more favorable for catalysis. KEY POINTS: • Multiple enzymatic properties of Penicillium oxalicum 16 BGL were coevolved. • A catalytically efficient triple mutant G414S/D421V/T441S was reported. • Molecular dynamics simulation supported the enhanced catalytic activity.

Co-evolution of ß-glucosidase activity and product tolerance for increasing cellulosic ethanol yield.

ß-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency ([Formula: see text]) for hydrolyzing pNPG and reduced strength of inhibition ([Formula: see text]) by glucose. To be specific, L-6C achieved a [Formula: see text] of 0.35 at a glucose concentration of 20 mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68 mM-1 s-1, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88 mM) to pNGP compared with 16BGL (1.69 mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL. We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.

Screening and Mutation of Saccharomyces cerevisiae UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature, Glucose and Ethanol.

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33 ± 1.34 to 122.22 ± 2.80 g/L at 30-45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18 ± 2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.

A Diagnostic Test Combining Molecular Testing with Phenotypic Pooled Antibiotic Susceptibility Improved the Clinical Outcomes of Patients with Non-E. coli or Polymicrobial Complicated Urinary Tract Infections.

Purpose: Complicated UTIs (cUTIs) cause significant morbidity and healthcare resource utilization and cost. Standard urine culture has limitations in detecting polymicrobial and non-E. coli infections, resulting in the under-diagnosis and under-treatment of cUTIs. In this study, patient-reported outcomes were compared between treated and untreated patients when an advanced diagnostic test combining multiplex-polymerase chain reaction (M-PCR) with a pooled antibiotic susceptibility method (P-AST) was incorporated into the patients' clinical management. Methods: Patients who had symptoms typical of cUTI and positive M-PCR/P-AST test results were recruited from urology clinics. Symptom reduction and clinical cure rates were measured from day 0 through day 14 using the American English Acute Cystitis Symptom Score (ACSS) Questionnaire. Clinical cure was defined based on the sum of the scores of four US Food and Drug Administration (FDA) symptoms and the absence of visible blood in the urine. Results: Of 264 patients with suspected cUTI, 146 (55.4%) had exclusively non-E. coli infections (115 treated and 31 untreated) and 190 (72%) had polymicrobial infections (162 treated and 28 untreated). Treated patients exhibited greater symptom reduction compared to untreated ones on day 14 for those with exclusively non-E. coli organisms (3.18 vs 1.64, p = 0.006) and polymicrobial infections (3.52 vs 1.41, p = 0.002), respectively. A higher percentage of treated patients than of untreated patients achieved clinical cure for polymicrobial infections on day 14 (58.7% vs 36.4%, p = 0.049). Conclusion: Patients with cUTIs treated based on the M-PCR/P-AST diagnostic test had significantly improved symptom reduction and clinical cure rates compared to untreated patients among those with non-E. coli or polymicrobial infections.

Multiplex Polymerase Chain Reaction/Pooled Antibiotic Susceptibility Testing Was Not Associated with Increased Antibiotic Resistance in Management of Complicated Urinary Tract Infections.

Objective: To compare antibiotic resistance results at different time points in patients with urinary tract infections (UTIs), who were either treated based upon a combined multiplex polymerase chain reaction (M-PCR) and pooled antibiotic susceptibility test (P-AST) or were not treated. Methods: The M-PCR/P-AST test utilized here detects 30 UTI pathogens or group of pathogens, 32 antibiotic resistance (ABR) genes, and phenotypic susceptibility to 19 antibiotics. We compared the presence or absence of ABR genes and the number of resistant antibiotics, at baseline (Day 0) and 5-28 days (Day 5-28) after clinical management in the antibiotic-treated (n = 52) and untreated groups (n = 12). Results: Our results demonstrated that higher percentage of patients had a reduction in ABR gene detection in the treated compared to the untreated group (38.5% reduction vs 0%, p = 0.01). Similarly, significantly more patients had reduced numbers of resistant antibiotics, as measured by the phenotypic P-AST component of the test, in the treated than in the untreated group (42.3% reduction vs 8.3%, p = 0.04). Conclusion: Our results with both resistance gene and phenotypic antibiotic susceptibility results demonstrated that treatment based upon rapid and sensitive M-PCR/P-AST resulted in reduction rather than induction of antibiotic resistance in symptomatic patients with suspected complicated UTI (cUTI) in an urology setting, indicating this type of test is valuable in the management of these types of patients. Further studies of the causes of gene reduction, including elimination of ABR gene-carrying bacteria and loss of ABR gene(s), are warranted.

Comparative Secretomics Analysis Reveals the Major Components of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30.

In this study, the major secretome components of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30 under wheat bran (WB) and rice straw (RS) solid-state fermentation were systematically analyzed. The activities of the major components, e.g., cellulase, hemicellulase, and amylase, were consistent with their abundance in the secretomes. P. oxalicum 16 secreted more abundant glycoside hydrolases than T. reesei RUT-C30. The main up-regulated proteins from the induction of WB, compared with that from RS, were amylase, pectinase, and protease, whereas the main down-regulated enzymes were cellulase, hemicellulase, swollenin, and lytic polysaccharide monooxygenase (LPMO). Specifically, WB induced more ß-1,4-glucosidases, namely, S8B0F3 (UniProt ID), and A0A024RWA5 than RS, but RS induced more ß-1,4-exoglucanases and ß-1,4-endoglucanases, namely, A0A024RXP8, A024SH76, S7B6D6, S7ZP52, A024SH20, A024S2H5, S8BGM3, S7ZX22, and S8AIJ2. The P. oxalicum 16 xylanases S8AH74 and S7ZA57 were the major components responsible for degrading soluble xylan, and S8BDN2 probably acted on solid-state hemicellulose instead of soluble xylan. The main hemicellulase component of T. reesei RUT-C30 in RS was the xyloglucanase A0A024S9Z6 with an abundance of 16%, but T. reesei RUT-C30 lacked the hemicellulase mannanase and had a small amount of the hemicellulase xylanase. P. oxalicum 16 produced more amylase than T. reesei RUT-C30, and the results suggest amylase S7Z6T2 may degrade soluble starch. The percentage of the glucoamylase S8B6D7 did not significantly change, and reached an average abundance of 5.5%. The major auxiliary degradation enzymes of P. oxalicum 16 were LPMOs S7Z716 and S7ZPW1, whereas those of T. reesei RUT-C30 were swollenin and LPMOs A0A024SM10, A0A024SFJ2, and A0A024RZP7.

Recombinant Penicillium oxalicum 16 ß-Glucosidase 1 Displays Comprehensive Inhibitory Resistance to Several Lignocellulose Pretreatment Products, Ethanol, and Salt.

ß-Glucosidase (BGL) is a rate-limiting enzyme of lignocellulose hydrolysis for second-generation bioethanol production, but its inhibition by lignocellulose pretreatment products, ethanol, and salt is apparent. Here, the recombinant Penicillium oxalicum 16 BGL 1 (rPO16BGL1) from Pichia pastoris GS115 kept complete activity at 0.2-1.4 mg/mL furan derivatives and phenolic compounds, 50 mg/mL sodium chloride (potassium chloride), or 100 mg/mL ethanol at 40 °C. rPO16BGL1 retained above 50% residual activity at 30 mg/mL organic acid sodium, and 60% residual activity at 40 °C with 300 mg/mL ethanol. Sodium chloride and potassium chloride had a complicated effect on rPO16BGL1, which resulted in activation or inhibition. The inhibition kinetics of the enzyme reaction demonstrated that organic acids and organic acid sodium were non-competitive inhibitors and that ethanol was a competitive inhibitor at < 1.5 mg/mL salicin. Moreover, substrate inhibition of the enzyme was found at > 2 mg/mL salicin, and the Km/KI and Km/KSI average values revealed that the inhibitory strength was ranked as salicin-organic acids > organic acids > salicin-organic acid sodium salt > organic acid sodium salt > salicin > salicin-KCl > salicin-NaCl > salicin-ethanol > ethanol.

Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3.

BACKGROUND: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low. RESULTS: In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20-30% improvement to that of the parental strain PC-3-7. CONCLUSIONS: This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Effects of cartilage polysaccharide on apoptosis of human hepatoma BEL-7402 cells and murine H22 hepatocarcinoma.

OBJECTIVE: To detect the mechanism of apoptosis on hepatoma BEL-7402 cells induced by cartilage polysaccharide in vitro and the effect on H22 hepatocarcinoma mice in vivo. MATERIALS AND METHODS: TUNEL assay, flow cytometry, immunofluorescent staining and animal experiments were used to examine the effects of cartilage polysaccharide on human hepatoma BEL-7402 cells in vitro or H22 hepatocarcinoma mice in vivo. RESULTS: We found that cartilage polysaccharide had a dramatic effect on inducing apoptosis in BEL-7402 cells in a dose-dependent and time-dependent manner. The molecular mechanism of the process of apoptosis was investigated, which was correlated with FAS-caspase and regulated the expression level of Vimentin and p21 protein. The survival rate and lifespan of the experimental group were better than for the model group. CONCLUSION: Our data demonstrate that cartilage polysaccharide may serve as a novel source of anti-hepatocarcinoma agent that may play an important role in future clinical trials.

Efficient (3R)-Acetoin Production from meso-2,3-Butanediol Using a New Whole-Cell Biocatalyst with Co-Expression of meso-2,3-Butanediol Dehydrogenase, NADH Oxidase, and Vitreoscilla Hemoglobin.

Acetoin (AC) is a volatile platform compound with various potential industrial applications. AC contains two stereoisomeric forms: (3S)-AC and (3R)-AC. Optically pure AC is an important potential intermediate and widely used as a precursor to synthesize novel optically active materials. In this study, chiral (3R)-AC production from meso-2,3-butanediol (meso-2,3-BD) was obtained using recombinant Escherichia coli cells co-expressing meso-2,3-butanediol dehydrogenase (meso-2,3-BDH), NADH oxidase (NOX), and hemoglobin protein (VHB) from Serratia sp. T241, Lactobacillus brevis, and Vitreoscilla, respectively. The new biocatalyst of E. coli/pET-mbdh-nox-vgb was developed and the bioconversion conditions were optimized. Under the optimal conditions, 86.74 g/l of (3R)-AC with the productivity of 3.61 g/l/h and the stereoisomeric purity of 97.89% was achieved from 93.73 g/l meso-2,3-BD using the whole-cell biocatalyst. The yield and productivity were new records for (3R)-AC production. The results exhibit the industrial potential for (3R)-AC production via whole-cell biocatalysis.

A Newly Isolated Penicillium oxalicum 16 Cellulase with High Efficient Synergism and High Tolerance of Monosaccharide.

Compared to Trichoderma reesei RUT-C30 cellulase (Trcel), Penicillium oxalicum 16 cellulase (P16cel) from the fermentation supernatant produced a 2-fold higher glucose yield when degrading microcrystalline cellulose (MCC), possessed a 10-fold higher ß-glucosidase (BGL) activity, but obtained somewhat lower other cellulase component activities. The optimal temperature and pH of ß-1,4-endoglucanase, cellobiohydrolase, and filter paperase from P16cel were 50-60 °C and 4-5, respectively, but those of BGL reached 70 °C and 5. The cellulase cocktail of P16cel and Trcel had a high synergism when solubilizing MCC and generated 1.7-fold and 6.2-fold higher glucose yields than P16cel and Trcel at the same filter paperase loading, respectively. Additional low concentration of fructose enhanced the glucose yield during enzymatic hydrolysis of MCC; however, additional high concentration of monosaccharide (especially glucose) reduced cellulase activities and gave a stronger monosaccharide inhibition on Trcel. These results indicate that P16cel is a more excellent cellulase than Trcel.

Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0.

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as 'LML 3.0', and an on-off control protocol of NHEJ pathway called 'OFN 1.0', using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.

Cloning, expression, sequence analysis, and partial characterization of two alkaline ß-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud.

Here we cloned and expressed two alkaline ß-1, 4-endoglucanases of Phaeosphaeria sp. LH21 from deep-sea mud. The two enzymes shared 71 and 63 % of identities with their known ß-1, 4-endoglucanases, respectively. According to the primary and spatial structures, the potential active sites of one of the two enzymes could be Asp122 and Asp11, while the other enzyme could be Asp16. The enzymatic properties of their recombinant enzymes from Pichia pastoris GS115 showed that they were optimally active at pH 8 and 60-65 °C, exhibited >90 % residual relative activities at pH 3-10, and obtained relative activities >75 % at pH 5-10.

Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning.

The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase). The crude CMCase only shows a 44.1-kDa band by activity staining after SDS-PAGE. It is optimally active at 55°C and pH 6.0, and is stable from pH 5.0-8.0 and at 45°C or below. The crude CMCase, which is not affected by Co(2+), is strongly activated in the presence of 10 mM Na(+), K(+), Ca(2+), Mg(2+), EDTA, and Mn(2+). It is strongly inhibited by 10 mM Fe(2+), Pb(2+), Al(3+), Zn(2+), Ag(+), Fe(3+), and Cu(2+). When compared with denim treatment by Novoprime A800 (a commercial neutral cellulase), crude CMCase exhibits a similar fabric weight loss and indigo dye removal. These results indicate that crude CMCase has potential application in denim biostoning.

A comparative study of ß-1, 4-endoglucanase (possessing ß-1, 4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3).

ß-1, 4-Endoglucanase (EG) from Bacillus subtilis LH was expressed in Escherichia coli Rosetta (DE3) and Pichia pastoris GS115, respectively. The CMCase activity of EG (EGE) from the cell lysate of DE3 reached 20,010U/ml, and that of EG (EGP) from the supernatant of GS115 was only 2008U/ml. EGE and EGP were bifunctional cellulases excluding ß-1, 4-glucosidase (BGL). The CMCases of them, optimally active at 65°C and pH 6.8, exhibited more than 80% residual activity at pH 5-10 and 60% activity at 40-70°C and pH 5-9. EGE (EGP) mixed with BGL had more than 1.5-fold higher CMCase and filter paperase activities compared to EGE (EGP). N-glycosylation protected EGP from immobilized-papain attack and accounted for 30kDa and a higher thermostability, whereas EGE was decomposed into a 33kDa active truncated EG (EGT) and two 18kDa fragments. EGE and EGP performed much better than EGT in denim biostoning.

Immunizing effects of cocultures of H22 hepatocarcinoma cells and cartilage polysaccharide on murine H22 hepatocarcinoma.

Hepatocarcinoma is a malignant cancer, which is threatening human lives. In order to disclose the immunizing effects of the cells and cartilage polysaccharide (CHCP) on liver cancer, murine H22 hepatocarcinoma model was set up. The survival time, life span, and survival rate of the CHCP group were better than model group or other groups which was immunized with cartilage short-chain polysaccharide (CPS) only or H22 cell lysate only. A series of experiments were proceeded and the results confirmed that the humoral immunity and cellular immunity were strengthened. HE staining, TUNEL assay, and Alcian staining were used to research the mechanism of the tumor specific antigen production which may be related to the apoptosis of H22 hepatocarcinoma cells induced by CPS or some new polysaccharide compound emerged. The animal experiment testified the relationship between H22 hepatocarcinoma cell apoptosis induced by CPS and the effect of murine H22 hepatocarcinoma immunoprophylaxis. Our data demonstrated that the coculture of cartilage polysaccharide and cancer cells may serve as a novel source of antihepatocarcinoma agent that may play an important role in future liver cancer immunoprophylaxis.

Resonance Raman analysis of nonlinear solvent dynamics: betaine-30 in ethanol.

Resonance Raman profiles for 14 vibrational modes of betaine-30 in ethanol at room temperature were measured at wavelengths within the first charge-transfer absorption band. The absorption spectrum and resonance Raman profiles were analyzed using time-dependent theory and a Brownian oscillator model modified to account for nonlinear solvent response; i.e., dependence of the solvent reorganization energy on the electronic state of the solute. As in our previous study of betaine-30 in acetonitrile, the solvent reorganization energy for the excited electronic state, determined from resonance Raman spectroscopy, was found to be smaller than that for the ground electronic state, determined from the absorption spectrum. The mode-dependent internal reorganization energies of betaine-30 in ethanol were found to be slightly larger than those of betaine-30 in acetonitrile. Temperature-dependent solvent reorganization energies for the ground electronic state were determined from analysis of the absorption line shape from 279 to 332 K and were found to decrease with increasing temperature. The influence of hydrogen bonding on the solvent and internal reorganization energy of betaine-30 is considered, and the physical basis for nonlinear solvent response is discussed.

Inertial solvent dynamics and the analysis of spectral line shapes: Temperature-dependent absorption spectrum of beta-carotene in nonpolar solvent.

The influence of solvent dynamics on optical spectra is often described by a stochastic model which assumes exponential relaxation of the time-correlation function for solvent-induced frequency fluctuations. In contrast, theory and experiment suggest that the initial (subpicosecond) phase of solvent relaxation, resulting from inertial motion of the solvent, is a Gaussian function of time. In this work, we employ numerical and analytical calculations to compare the predicted absorption line shapes and the derived solvent reorganization energies obtained from exponential (Brownian oscillator) versus Gaussian (inertial) solvent dynamics. Both models predict motional narrowing as the ratio kappa = Lambda/Delta is increased, where Lambda and Delta are the frequency and variance, respectively, of the solvent-induced frequency fluctuations. However, the motional narrowing limit is achieved at lower values of kappa for the Brownian oscillator model compared to the inertial model. For a given line shape, the derived value of the solvent reorganization energy lambdasolv is only weakly dependent on the solvent relaxation model employed, though different solvent parameters Lambda and Delta are obtained. The two models are applied to the analysis of the temperature-dependent absorption spectrum of beta-carotene in isopentane and CS2. The derived values of lambdasolv using the Gaussian model are found to be in better agreement with the high temperature limit of Delta2/2kBT than are the values obtained using the Brownian oscillator model. In either approach, the solvent reorganization energy is found to increase slightly with temperature as a result of an increase in the variance Delta of the solvent-induced frequency fluctuations.