Using three-dimensional modelling of the anterior sclera to investigate the scleral profile in myopic eyes.

PURPOSE: This study used three-dimensional (3D) modelling to investigate scleral profiles in myopic eyes and compare them with emmetropic eyes. METHODS: In this prospective observational study, the eyes of 151 participants were analysed using the corneoscleral profile module (CSP) of the Pentacam HR. Non-rotationally symmetrical ellipsoids were fitted to the anterior scleral sagittal height. Three radii were analysed, namely the nasal-temporal (Rx), superior-inferior (Ry) and anterior-posterior (Rz) orientations. Additionally, the area index (AI) and aspherical parameters (Qxy, Qxz and Qyz) of the anterior sclera-fitted ellipsoid (ASFE) were quantified. RESULTS: The findings showed an increase in Rx (-0.349 mm/D), Ry (-0.373 mm/D), Rz (-1.232 mm/D) and AI (-36.165 mm2 /D) with increasing myopia. From emmetropia to high myopia, the vertical and horizontal planes of the anterior sclera became increasingly prolate (emmetropia, Qxz: 0.02, Qyz: 0.01; low myopia, Qxz: -0.28, Qyz: -0.28; high myopia, Qxz: -0.41, Qyz: -0.43). There were no significant differences in the coronal plane across the three groups (H = 2.65, p = 0.27). The anterior scleral shape of high myopes in the horizontal and vertical planes was more prolate than that of emmetropes and low myopes (Qxz, high myopes vs. low myopes: p = 0.03, high myopes vs. emmetropes: p < 0.001; Qyz, high myopes vs. low myopes: p = 0.04, high myopes vs. emmetropes: p < 0.001). CONCLUSIONS: As the degree of myopia increased, non-uniform anterior scleral enlargement was observed. These findings provide a better understanding of the anterior segment with varying degrees of myopia.

Trajectories of spherical equivalent refraction from grades 1 to 4 in Chinese children.

BACKGROUND: The development trajectories of children's SER remain unknown. This study aimed to characterize spherical equivalent refraction (SER) trajectories during grades 1-4 in Chinese children. METHODS: This prospective cohort study included 1226 first-grade non-myopic children from 12 public primary schools, randomly selected in two districts in Guangzhou, China. From November 2018 to March 2022, four-wave ocular examinations and questionnaire surveys have been completed. The group-based trajectory modeling was used to explore SER trajectories in grades 1-4. RESULTS: All five trajectories showed an upward trend and rose faster after grade 2. Children in the sharp-developing (n = 44), high-developing (n = 136), and rapid-developing (n = 237) myopia groups developed myopia before grades 2, 3, and 4, respectively. Their SER development speed remained at a relatively high level after myopia, almost consistent with that before myopia. Children in the moderate-developing (n = 418) and low-developing (n = 391) non-myopia groups did not develop myopia before grade 4. Some characteristics in grade 1 were independently associated with SER trajectories, including sex, axial length, number of parents with myopia, residence, academic achievement, and the duration of outdoor activity. Based on the baseline characteristics, we established the model predicting the probability of children belonging to each group. CONCLUSIONS: Myopia interventions are best started in grade 1 or preschool age. If interventions are not taken in time, the latest intervention window might be in grades 1, 2, and 3 for children with a high probability of belonging to the sharp-developing, high-developing, and rapid-developing myopia groups, respectively. The above probabilities might be predicted using the model we established. Moreover, the interventions for myopic children shouldn't be ignored.

Axial length shortening in myopic children with Stickler syndrome after repeated low-level red-light therapy.

AIM: To report the myopia-controlling effect of repeated low-level red-light (RLRL) therapy in patients with Stickler syndrome (STL), an inherited collagenic disease typically presenting with early onset myopia. METHODS: Three STL children, aged 3, 7, and 11y, received RLRL therapy throughout the follow-up period of 17, 3, and 6mo, respectively after exclusion of fundus anomalies. Data on best-corrected visual acuity (BCVA), intraocular pressure, cycloplegic subjective refraction, ocular biometrics, scanning laser ophthalmoscope, optical coherence tomography, genetic testing, systemic disease history, and family history were recorded. RESULTS: At the initiation of the RLRL therapy, the spherical equivalent (SE) of 6 eyes from 3 patients ranged from -3.75 to -20.38 D, axial length (AL) were from 23.88 to 30.68 mm, and BCVA were from 0.4 to 1.0 (decimal notation). Myopia progression of all six eyes slowed down after RLRL therapy. AL in five out of the six eyes shortened -0.07 to -0.63 mm. No side effects were observed. CONCLUSION: Three cases of STL whose progression of myopic shift and AL elongation are successfully reduced and even reversed after RLRL therapy.

Cold-Adapted Proteases: An Efficient and Energy-Saving Biocatalyst.

The modern biotechnology industry has a demand for macromolecules that can function in extreme environments. One example is cold-adapted proteases, possessing advantages such as maintaining high catalytic efficiency at low temperature and low energy input during production and inactivation. Meanwhile, cold-adapted proteases are characterised by sustainability, environmental protection, and energy conservation; therefore, they hold significant economic and ecological value regarding resource utilisation and the global biogeochemical cycle. Recently, the development and application of cold-adapted proteases have gained gaining increasing attention; however, their applications potential has not yet been fully developed, which has seriously restricted the promotion and application of cold-adapted proteases in the industry. This article introduces the source, related enzymology characteristics, cold resistance mechanism, and the structure-function relationship of cold-adapted proteases in detail. This is in addition to discussing related biotechnologies to improve stability, emphasise application potential in clinical medical research, and the constraints of the further developing of cold-adapted proteases. This article provides a reference for future research and the development of cold-adapted proteases.

The Causal Effect of Education on Myopia: Evidence That More Exposure to Schooling, Rather Than Increased Age, Causes the Onset of Myopia.

Purpose: To distinguish the effects of age and grade on the development of myopia. Methods: Grade 1 (n = 1465, mean age 6.71 ± 0.29 years; 53.5% male) and Grade 2 students (n = 1381, mean age 7.76 ± 0.30 years; 52.5% male) were examined in 2018, with a follow-up examination in 2019. Cycloplegic spherical equivalent (SE) in diopter (D) was measured. Regression discontinuity (RD) analysis was used to assess the causal effects on refraction at each visit. Results: The sample in a grade was divided into three 4-month age blocks according to their birth month, the youngest, middle, and the oldest. At the 2018 visit, within each grade, there were no significant differences in SE among age blocks (all P > 0.05), despite an age range of 12 months. However, comparing the youngest block in Grade 2 to the oldest block in Grade 1, an average age difference of four months, a significant difference in SE was found (0.82 ± 0.69 D vs. 1.05 ± 0.55 D, t-test P < 0.01). Formal RD analysis found a significant casual effect of grade increase on myopic refraction shift (ß = -0.32 D; 95% CI, -0.73 to -0.01; P = 0.042). Consistent results were found using the 2019 data. Conclusions: Increased grade, rather than increasing age, is the major cause of myopic shifts in refraction. A causal link implies that interventions aimed at reducing the myopigenic exposures experienced during a school year have the potential to markedly reduce the myopic shifts in refraction associated with a grade of schooling.

A novel thermostable and salt-tolerant carboxylesterase involved in the initial aerobic degradation pathway for pyrethroids in Glycomyces salinus.

The long-term and excessive use of pyrethroid pesticides poses substantial health risks and ecosystem concerns. Several bacteria and fungi have been reported that could degrade pyrethroids. The ester-bond hydrolysis using hydrolases is the initial regulatory metabolic reaction of pyrethroids. However, the thoroughly biochemical characterization of hydrolases involved in this process is limited. Here, a novel carboxylesterase, designated as EstGS1 that could hydrolyze pyrethroid pesticides was characterized. EstGS1 showed low sequence identity (<27.03%) compared to other reported pyrethroid hydrolases and belonged to the hydroxynitrile lyase family that preferred short short-chain acyl esters (C2 to C8). EstGS1 displayed the maximal activity of 213.38 U/mg at 60 °C and pH 8.5 using pNPC2 as substrate, with Km and Vmax were 2.21 ± 0.72 mM and 212.90 ± 41.78 µM/min, respectively. EstGS1 is a halotolerant esterase and remains stable in 5.1 M NaCl. Based on molecular docking and mutational analysis, the catalytic triad of S74-D181-H212 and three other substrate-binding residues I108, S159, and G75 are critical for the enzymatic activity of EstGS1. Additionally, 61 and 40 mg/L of deltamethrin and λ-cyhalothrin were hydrolyzed by 20 U of EstGS1 in 4 h. This work presents the first report on a pyrethroid pesticide hydrolase characterized from a halophilic actinobacteria.

Long-term effect of orthokeratology on choroidal thickness and choroidal contour in myopic children.

PURPOSE: To investigate the long-term effect of orthokeratology (ortho-k) on the choroidal thickness and choroidal contour in myopic children. METHODS: Subjects were from a conducted 2-year randomised clinical trial. Children (n=80) aged 8-12 years with spherical equivalent refraction of -1.00 to -6.00D were randomly assigned to the control group (n=40) and ortho-k group (n=40). Optical coherence tomography images were collected at the baseline, 1-month, 6-month, 12-month, 18-month and 24-month visits, then the choroidal thickness and choroid contour were calculated. Axial length (AL) and other ocular biometrics were also measured. RESULTS: During 2 years, in the control group, the choroidal thickness became thinning and the choroidal contour became prolate with time at all visits (all p<0.001). Ortho-k can improve the choroidal thickness (all p<0.001) and maintain the choroidal contour at all visits (all p<0.05). In the ortho-k group, the choroidal contour was less changed in the temporal than nasal (p=0.008), and the choroidal thickness was more thickening in the temporal 3 mm (p<0.001). Two-year change in choroidal thickness was significantly associated with the 2-year AL change in the control group (r=-0.52, p<0.001), however, this trend was broken by ortho-k (r=-0.05, p=0.342). After being adjusted by other variables in the multivariable regression model, the effect of ortho-k on choroidal thickness was stable. CONCLUSIONS: In the current 2-year prospective study, ortho-k can improve the choroidal thickness and maintain the choroidal contour, but this effect diminished in a long term. Further study with larger sample size and longer follow-up is warranted to refine this issue.

Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance.

Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment.

Biochemical and Molecular Characteristics of a Novel Hyaluronic Acid Lyase from Citrobacter freundii.

The Gram-negative strain of Citrobacter freundii, YNLX, has the ability to degrade hyaluronic acid. In this study, we expressed a C. freundii hyaluronic acid lyase, from polysaccharide lyase family 8, in Escherichia coli. The purified recombinant enzyme (rHynACF8) showed a substantially higher cleavage activity of hyaluronic acid than chondroitin sulfate. We found that its optimal pH and temperature are 5.5 and 35 °C, respectively. In addition, the enzyme activity was not notably affected by most metal ions. Km and kcat of rHynACF8 towards HA were 1.5 ± 0.01 mg/mL and 30.9 ± 0.5 /s, respectively. rHynACF8 is an endo-acting enzyme. Its cleavage products had dramatically increased antioxidant activity than hyaluronic acid in vitro (p < 0.001). As the molecular weight of hyaluronic acid decreased, the intramolecular interactions among antioxidant functional groups were removed; in the process of the cracking reaction, new double bonds formed and conjugated with the carbonyl group. We presumed that the structural change is the critical factor influencing antioxidant capacity. Overall, we found that rHynACF8 from Gram-negative bacteria with metal ion resistance, indicated the relationship between the function and structure of its antioxidant cleavage product.

Rates of Myopia Development in Young Chinese Schoolchildren During the Outbreak of COVID-19.

Importance: During the outbreak of COVID-19, outdoor activities were limited and digital learning increased. Concerns have arisen regarding the impact of these environmental changes on the development of myopia. Objective: To investigate changes in the development of myopia in young Chinese schoolchildren during the outbreak of COVID-19. Design, Setting, and Participants: In this observational study, 2 groups of students from 12 primary schools in Guangzhou, China, were prospectively enrolled and monitored from grade 2 to grade 3. Comparisons between the exposure and nonexposure groups were made to evaluate any association between environmental changes during the COVID-19 outbreak period and development of myopia. The exposure group received complete eye examinations in November and December 2019 and November and December 2020. The nonexposure group received examinations in November and December 2018 and November and December 2019. Main Outcomes and Measures: Changes in cycloplegic spherical equivalent refraction (SER), axial length (AL) elongation, and myopia incidence from grade 2 to grade 3. Results: Among the 2679 eligible students in grade 2 (mean [SD] age, 7.76 [0.32] years; 1422 [53.1%] male), 2114 (1060 in the nonexposure group and 1054 in the exposure group) were reexamined in grade 3. Compared with the period from November and December 2018 to November and December 2019, the shift of SER, AL elongation, and myopia incidence from grade 2 to grade 3 from November and December 2019 to November and December 2020 was 0.36 D greater (95% CI, 0.32-0.41; P < .001), 0.08 mm faster (95% CI, 0.06-0.10; P < .001), and 7.9% higher (95% CI, 5.1%-10.6%; P < .001), respectively. In grade 3 students, the prevalence of myopia increased from 13.3% (141 of 1060 students) in November and December 2019 to 20.8% (219 of 1054 students) in November and December 2020 (difference [95% CI], 7.5% [4.3-10.7]; P < .001); the proportion of children without myopia and with SER greater than -0.50 D and less than or equal to +0.50 D increased from 31.1% (286 of 919 students) to 49.0% (409 of 835 students) (difference [95% CI], 17.9% [13.3-22.4]; P < .001). Conclusions and Relevance: In this study, development of myopia increased during the COVID-19 outbreak period in young schoolchildren in China. Consequently, myopia prevalence and the proportion of children without myopia who were at risk of developing myopia increased. Future studies are needed to investigate long-term changes in myopia development after the COVID-19 pandemic.

Analysis of Saccharification Products of High-Concentration Glutinous Rice Fermentation by Rhizopus nigricans Q3 and Alcoholic Fermentation of Saccharomyces cerevisiae GY-1.

A two-stage process was used to prepare rice alcohol, i.e., saccharification of glutinous rice by Rhizopus nigricans Q3, followed by Saccharomyces cerevisiae's fermentation. Rhizopus nigricans Q3 was cultured during the saccharification stage, and Saccharomyces cerevisiae GY-1 was added in the fermentation stage. Total sugar content and reducing sugar content in these two stages were analyzed. The relationship between the production proportion and consumption of the reducing sugar in the saccharification interval was analyzed using reducing sugar indices. It is an important rule that the high-concentration syrup and oligosaccharides prepared by glutinous rice could reach 42°Bx and 250 mg/mL by high-concentration fermentation in the growth stage of R. nigricans Q3.

Enzymatic preparation of manno-oligosaccharides from locust bean gum and palm kernel cake, and investigations into its prebiotic activity

BACKGROUND: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. RESULTS: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. CONCLUSIONS: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum, which would have potential application in the biotechnological applications.

[Exploration and practice in ideological education in the course of "Protein and Enzyme Engineering"].

Protein and Enzyme Engineering is the core and required course for colleague students majored in biotechnology, which plays an important role in the professional training system. In accordance with the "Guidelines for the Development of Ideological Education in Higher Education Institutions" issued by the Ministry of Education, we explored the combination of course teaching with ideological education by considering the features of the biotechnology major and the course and setting up rational teaching objectives. This paper described the strategy, design, implementation and evaluation approaches that were used in the course of "Protein and Enzyme Engineering" to achieve a good integration. The practice starts from story-telling, discussion of life, case study, hot issues discussion, literature discussion and presentations. The scientific spirits, civic character, global vision, eco-civilization and legal consciousness, as well as their native land emotion and cultural confidence, were boosted. The natural integration of the ideological education into the whole process of this course helped to better achieve the goal of curriculum education while promoting teaching excellence.

Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2.

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure's stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

Improving low-temperature activity and thermostability of exo-inulinase InuAGN25 on the basis of increasing rigidity of the terminus and flexibility of the catalytic domain.

Enzymes displaying high activity at low temperatures and good thermostability are attracting attention in many studies. However, improving low-temperature activity along with the thermostability of enzymes remains challenging. In this study, the mutant Mut8S, including eight sites (N61E, K156R, P236E, T243K, D268E, T277D, Q390K, and R409D) mutated from the exo-inulinase InuAGN25, was designed on the basis of increasing the number of salt bridges through comparison between the low-temperature-active InuAGN25 and thermophilic exo-inulinases. The recombinant Mut8S, which was expressed in Escherichia coli, was digested by human rhinovirus 3 C protease to remove the amino acid fusion sequence at N-terminus, producing RfsMut8S. Compared with wild-type RfsMInuAGN25, the mutant RfsMut8S showed (1) lower root mean square deviation values, (2) lower root mean square fluctuation (RMSF) values of residues in six regions of the N and C termini but higher RMSF values in five regions of the catalytic pocket, (3) higher activity at 0-40°C, and (4) better thermostability at 50°C. This study proposes a way to increase low-temperature activity along with a thermostability improvement of exo-inulinase on the basis of increasing the rigidity of the terminus and the flexibility of the catalytic domain. These findings may prove useful in formulating rational designs for increasing the thermal performance of enzymes.

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25.

Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90-95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli. After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation-π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus-function relationships that are useful for rational design of thermostability of exo-inulinases.

Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract.

The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointestinal tract microbe. By screening the fecal metagenomic library, 48 salt-tolerant clones were detected, of which 10 salt-tolerant clones exhibited stronger tolerance to 7% (wt/vol) NaCl and stability in different concentrations of NaCl [5%-9% (wt/vol)]. High-throughput sequencing and biological information analysis showed that 91 potential genes encoded proteins and enzymes that were widely involved in salt tolerance. Furthermore, two trehalose-6-phosphate hydrolase genes, namely, tre_P2 and tre_P3, were successfully cloned and expressed in Escherichia coli BL21 (DE3). By virtue of the substrate of p-nitrophenyl-α-D-glucopyranoside (pNPG) which can be specifically hydrolyzed by trehalose-6-phosphate hydrolase to produce glucose and p-nitrophenol, the two enzymes can act optimally at pH 7.5 and 30°C. Steady-state kinetics with pNPG showed that the K M and K cat values were 15.63 mM and 10.04 s-1 for rTRE_P2 and 12.51 mM and 10.71 s-1 for rTRE_P3, respectively. Characterization of enzymatic properties demonstrated that rTRE_P2 and rTRE_P3 were salt-tolerant. The enzymatic activity increased with increasing NaCl concentration, and the maximum activities of rTRE_P2 and rTRE_P3 were obtained at 4 and 3 M NaCl, respectively. The activities of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 M NaCl. This study is the first to report the identification as well as molecular and biochemical characterization of salt-tolerant trehalose-6-phosphate hydrolase from the metagenomic library of the gastrointestinal tract. Results indicate the existence of numerous salt-tolerant genes and enzymes in gastrointestinal microbes and provide new insights into the salt-tolerant mechanisms in the gastrointestinal environment.

Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli.

BACKGROUND: Phthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation. RESULTS: A carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC2 as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD600 over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min. CONCLUSIONS: This work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy.

Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.

Research Article Product Composition Analysis and Process Research of Oligosaccharides Produced from Enzymatic Hydrolysis of High-Concentration Konjac Flour.

There is a huge variability in reducing sugars, viscosity, and composition of oligosaccharides in the hydrolyzed products of konjac flour with different concentrations. We analyzed the factors affecting reducing sugars, viscosity, and the average degree of polymerization (DP) during the preparation of oligosaccharides from konjac flour hydrolyzed by ß-mannanase under the high-concentration solute hydrolysis model. Hydrolysate of konjac flour, using concentrations ranging from 50 to 200 g/L, was directly added into 20 U/mL of ß-mannanase solution. The results showed that when the proportion of the water content in the solution decreased, the viscosity of the solution and the DP of polysaccharides changed significantly. When the viscosity of the hydrolysate was controlled within the range of 30-20 mPa·s, the concentration of the reducing sugars was maintained in the range of 9-13 g/L and the average DP of the polysaccharides was controlled in the range of 2.42-9.78. We also found that a high concentration of hydrolysate was beneficial for decreasing the production of reducing sugars, and the diversification of macromolecular glycan was beneficial to the preparation of functional sugars. Moreover, we observed that the proportion of reducing sugars with free water content was high and that the preparation of oligosaccharides via the high-concentration solid-state method increased product diversity.