CDK12 Promotes the Proliferation, Migration, and Angiogenesis of Gastric Carcinoma via Activating the PI3K/AKT/mTOR Signaling Pathway.

Cyclin-dependent kinase 12 (CDK12) has been found to regulate tumor progression. However, its function in gastric carcinoma (GC) remains controversial. This work aimed to explore the exact effect of CDK12 on GC progression. We detected the expression of CDK12 in GC cells and normal gastric mucosal epithelial cells. Then CDK12 function on GC cell proliferation, migration, and angiogenesis was researched by colony formation experiment, Transwell experiment, and angiogenesis assay. Moreover, CDK12 effect on the PI3K/AKT/mTOR pathway activity was explored by western blot. Further, we used LY294002 (10 µM) to treat GC cells to verify whether CDK12 regulates GC progression by activating the PI3K/AKT/mTOR pathway. Additionally, CDK12 effect on the expression of prognostic factors of GC was detected by western blot, including alkaline phosphatase (ALP) and Ki67. Quantitative real-time polymerase chain reaction and western blot were utilized to evaluate the expression of mRNAs and proteins. As a result, CDK12 was upregulated in GC cells. CDK12 overexpression facilitated the proliferation, migration, and angiogenesis of GC cells. However, CDK12 silencing showed an opposite result. CDK12 overexpression activated the PI3K/AKT/mTOR pathway, but CDK12 silencing inactivated it in GC cells. The blockage of the PI3K/AKT/mTOR pathway induced by LY294002 treatment counteracted the promotion of CDK12 on the proliferation, migration, and angiogenesis of GC. Further, CDK12 silencing suppressed the expression of ALP and Ki67 proteins in GC cells. Taken together, CDK12 promotes the proliferation, migration, and angiogenesis of GC by activating the PI3K/AKT/mTOR pathway. It may be a novel target for GC treatment.

Modification and application of highly active alkaline pectin lyase.

Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.

Room-Temperature Phosphorescence with Variable Lifetime of Halogen-Comprising Coordination Polymers.

Materials with lifetime-tunable room-temperature phosphorescence are fascinating for multiple encryption-decryption security applications. Herein, by introducing different halogen ions, that is, Cl, Br, and I, together with organic luminescent units to bond to a zinc center, three coordination polymers were hydro(solvo)thermally synthesized. The results show that three coordination polymers present room-temperature phosphorescence with different lifetimes. Furthermore, a multiple encryption-decryption system combining temporal and spatial resolution characteristics was designed.

Enhanced Room-Temperature Phosphorescence of an Organic Ligand in 3D Hybrid Materials Assisted by Adjacent Halogen Atom.

Room-temperature phosphorescence (RTP) materials have potential applications in anticounterfeiting, biochemistry, and optical recording. It is significant to develop a novel approach to enriching RTP materials. Here two new hybrid RTP solids were obtained by the introduction of organic 1,3,5-tris(1-imidazolyl)benzene and halogen atoms to simultaneously coordinate with inorganic moieties. Single-crystal X-ray analysis reveals that Br atoms are oriented toward the adjacent organic molecules that can play an important heavy-atom effect (HAE). Notably, such a HAE realized by orienting the Br atom to the location immediately adjacent to the triplets produced can efficiently facilitate intersystem crossing and results in RTP with intensity similar to that of the fluorescence in steady-state photoluminescence.

Room-Temperature Phosphorescence with Excitation-Energy Dependence and External Heavy-Atom Effect in Hybrid Zincophosphites.

Room-temperature phosphorescence (RTP) materials have gained much attention, because of their applications in chemical sensing, optoelectronics, and security systems. Transition-metal complexes, particularly those of IrIII, PtII, RuII, and AuI, have preciously been investigated in the quest for excellent RTP materials. Recently, the pure organic molecules caught the eyes of researchers. Although great achievement has been reached, expanding the available types of RTP materials and hunting for top-performing RTP materials are still significant to promote the development of RTP materials. In this work, we report a series of isostructural hybrid zincophosphite [Zn3(HPO3)2(tib)2]X2 (X- = Cl-, Br-, I-; tib = 1,3,5-tris(1-imidazolyl)-benzene), which feature a cationic host structure and an anionic guest (X-). Because of the restriction of molecular vibrations/rotations of organic luminogens (tib) and the heavy-atom effect of the guest halide ion (X-), the title compounds exhibit almost pure RTP with absolute phosphorescence quantum yields of 5.8%-9.1%. More interestingly, unique excitation-energy-dependent phosphorescence has been observed in these hybrid materials. The phosphorescence origin has also been illustrated by theoretical calculations. Our work provides new insights into the design of RTP materials. Considering the structural diversity together with the rich host-guest chemistry of metal-phosphite/phosphate, we offer a new avenue to explore superior crystalline RTP materials.

Structural impairment patterns in peripapillary retinal fiber layer and retinal ganglion cell layer in mitochondrial optic neuropathies.

AIM: To evaluate the structural injure patterns in peripapillary retinal fiber layer (pRNFL), retinal ganglion cell layer (RGCL) and their correlations to visual function in various mitochondrial optic neuropathies (MON) to offer help to their differential diagnosis. METHODS: Totally 32 MON patients (60 eyes) were recruited within 6mo after clinical onsets, including 20 Leber hereditary optic neuropathy (LHON) patients (37 eyes), 12 ethambutol-induced optic neuropathy (EON) patients (23 eyes), and 41 age-gender matched healthy controls (HC, 82 eyes). All subjects had pRNFL and RGCL examinations with optic coherence tomography (OCT) and visual function tests. RESULTS: In the early stages of MON, the temporal pRNFL thickness decreased (66.09±22.57 µm), but increased in other quadrants, compared to HC (76.95±14.81 µm). The other quadrants remaining stable for LHON and EON patients besides the second hour sector of pRNFL thickness reduced and the temporal pRNFL decreased (56.78±15.87 µm) for EON. Total macular thickness in MON reduced remarkably (279.25±18.90 µm; P=0.015), which mainly occurring in the inner circle (3 mm diameter of circle) and the nasal temporal sectors in the outer circle (5.5 mm diameter of circle), in contrast to those in HC. RGCL thickness reduced in each sector of the macula (61.90±8.73 µm; P≤0.001). It strongly showed the correlationship of best corrected visual acuity (R=0.50, P=0.0003) and visual field injury (R=0.54, P=0.0002) in MON patients. CONCLUSION: OCT is a potential tool for detecting structural alterations in the optic nerves of various MON. Different types of MON may have different damage patterns.

Thoracolumbar Intervertebral Disc Area Morphometry in Elderly Chinese Men and Women: Radiographic Quantifications at Baseline and Changes at Year-4 Follow-up.

STUDY DESIGN: A population-based radiographic study with longitudinal follow-up. OBJECTIVE: To develop a quantitative index for lumbar disc space narrowing (DSN) evaluation in elderly subjects; to determine how DSN in the elderly is influenced by osteoporosis and sex. SUMMARY OF BACKGROUND DATA: There is paucity of research on quantitative classification of lumbar DSN based on disc areal morphometry. METHODS: With the database of Osteoporotic Fractures in Men (Hong Kong) and Osteoporotic Fractures in Women (Hong Kong) Studies and those who attended the year-4 follow-up (n = 1519 for men and n = 1546 for women), data of 491 women and 592 men were randomly selected. The anterior, middle, and posterior heights; anteroposterior diameter; and area of intervertebral discs (T4T5 to L4L5) were measured on lateral radiographs. Disc area index for lumbar spine (DAIL, disc area divided by the mean of the sum of square of the adjacent upper and lower vertebrae mid-height anterior-posterior diameter) was developed and compared with semiquantitative DSN expert grading. RESULTS: DAIL correlated with semiquantitative grading, with sensitivity and specificity varying from 87.3% to 96.8% for grade 1 DSN (<30% reduction in disc height), and 92.9% to 100% for grade 3 DSN (>60% reduction in disc height). The thoracolumbar disc area loss among men and women during 4-years' follow-up period varied between 1.32% and 3.56%, and it was greater for women (mean: 2.44%) than for men (mean: 1.90%, P = 0.044). Majority of lumbar DSN progressions during 72 to 76 years old were progression from normal disc space to grade 1 DSN. Osteoporosis was associated with greater disc area decrease, both for thoracic and lumbar discs. CONCLUSION: Lumbar DSN can be quantified using DAIL. In elderly Chinese, intervertebral disc narrowing over a 4-year period was greater in women than men, and associated with the presence of osteoporosis. LEVEL OF EVIDENCE: 3.

Decompressive Craniectomy With Bifrontal Coronal Incision in the Management of Fronto-Temporal Contusion and Laceration for Early Cranioplasty.

The present study aims to explore the effectiveness of decompressive craniectomy with bifrontal coronal incision in the management of severe contusion and laceration of bilateral fronto-temporal lobes, as well as the outcomes of early cranioplasty. The authors performed the bifrontal decompressive craniectomy on 56 patients with contusion and laceration of bilateral frontal and temporal lobes, and their follow-up treatment outcomes were tracked within 6 months using Glasgow Outcome Scale. The results showed that 33 patients (out of 56, 58.9%) have recovered, 12 patients (out of 56, 21.4%) have moderate defects, 5 patients (out of 56, 8.9%) have severe defects, 3 patients (out of 56, 5.3%) stayed in persistent vegetative status, and the remaining 3 patients (out of 56, 5.3%) have been dead. There was no persistent temporal hollowing. No patients required revision surgery with modified titanium mesh in this study. Particularly, 28 patients have successfully accepted the early cranioplasty with bone flap or computer-assisted design titanium mesh, and showed good recovery. These results together indicated that the decompressive craniectomy with bifrontal coronal incision in the management of severe contusion and laceration of bilateral fronto-temporal lobes can significantly relieve the comorbidity of intracranial hypertension, and improve the prognosis obviously, thus finally increasing the probability of successful rescue and decreasing the probability of mortality and disability.

Retinal segmented layers with strong aquaporin-4 expression suffered more injuries in neuromyelitis optica spectrum disorders compared with optic neuritis with aquaporin-4 antibody seronegativity detected by optical coherence tomography.

PURPOSE: To evaluate retinal segmented layer alterations in optic neuritis (ON) in an AQP4-Ab seropositive (AQP4-Ab+/ON) cohort and in neuromyelitis optica (NMO) with ON eyes (NMO-ON) compared with an AQP4-Ab seronegative ON (AQP4-Ab-/ON) cohort using optical coherence tomography (OCT). METHODS: We recruited 109 patients with ON (161 eyes) and 47 healthy controls. All patients with ON were subdivided into three subcohorts: 37 patients (54 eyes) with AQP4-Ab+/ON, 45 patients (65 eyes) with AQP4-Ab-/ON and 27 patients (42 eyes) with NMO-ON. All subjects were evaluated for their peripapillary retinal nerve fibre layer (pRNFL) and inner macular segmented layer using OCT. RESULTS: AQP4-Ab+/patients with ON had the same structural injury patterns as patients with NMO-ON, and the injury patterns were distinct from those of AQP4-Ab-/patients with ON. NMO-ON and AQP4-Ab+/ON preferentially damaged the pRNFL (all p=0.000), the macular retinal nerve fibre layer (mRNFL; p=0.000 and 0.032, respectively), and the inner plexiform layer (IPL; p=0.000 and 0.006, respectively) without differences in the retinal ganglion cell layer (p=0.106 and 0.374, respectively) compared with AQP4-Ab-/patients with ON. The thickness of the inner nuclear layer (INL) increased in NMO-ON (p=0.043) compared with that of AQP4-Ab-/ON without a significant difference in AQP4-Ab+/ON versus AQP4-Ab-/ON (p=0.353). When the thickness of the inferior nasal quadrant (NI) of the pRNFL was reduced to ≤46.5 µm (area under the curve 0.772, sensitivity 89.2% and specificity 57.5%) 6 months after ON onset, NMO was considered. CONCLUSIONS: AQP4-Ab+/ON produced similar structural injury patterns as NMO-ON. The pRNFL, mRNFL and IPL in the two types of ON and the INL in NMO-ON suffered more damage than those in AQP4-Ab-/ON, which could be associated with strong aquaporin-4 expression. The thickness of the NI of the pRNFL could be a potential clue for predicting ON progression to definite NMO.

Increased low back pain prevalence in females than in males after menopause age: evidences based on synthetic literature review.

Female sex hormones play an important role in the etiology and pathophysiology of a variety of musculoskeletal degenerative diseases. Postmenopausal women show accelerated disc degeneration due to relative estrogen deficiency. This literature review aims to validate or falsify this hypothesis, i.e., while overall females have higher prevalence of low back pain (LBP) across all age groups, this male vs. female difference in LBP prevalence further increases after female menopause age. The literature search was performed on PubMed on January 2, 2016. The search word combination was (low back pain) AND prevalence AND [(males OR men) AND (females OR women)]. The following criteria were taken to include the papers for synthetic analysis: (I) only English primary literatures on nonspecific pain; (II) only prospective studies on general population, but not population with occupational LBP causes, of both males and female subjects studied using the same LBP criterion, ages-specific information available, and males and female subjects were age-matched; (III) studies without major quality flaws. In total 98 studies with 772,927 subjects were analyzed. According to the information in the literature, participant subjects were divided into four age groups: (I) school age children group: 6-19 years; (II) young and middle aged group: 20-50 years; (III) mixed age group: data from studies did not differentiate age groups; (IV) elderly group: ≥50 years old. When individual studies were not weighted by participant number and each individual study is represented as one entry regardless of their sample size, the median LBP prevalence ratio of female vs. males was 1.310, 1.140, 1.220, and 1.270 respectively for the four age groups. When individual studies were weighted by participant number, the LBP prevalence ratio of female vs. males was 1.360, 1.127, 1.185, and 1.280 respectively for the four groups. The higher LBP prevalence in school age girls than in school age boys is likely due to psychological factors, female hormone fluctuation, and menstruation. Compared with young and middle aged subjects, a further increased LBP prevalence in females than in males was noted after menopause age.

Therapeutic effects of metformin and laparoscopic ovarian drilling in treatment of clomiphene and insulin-resistant polycystic ovary syndrome.

OBJECTIVE: To compare the therapeutic effects of metformin (Met) and laparoscopic ovarian drilling (LOD) in clomiphene and insulin-resistant patients with polycystic ovary syndrome (CIRPCOS). METHODS: A total of 110 patients were randomly divided into two groups. One group was administered Met (n = 55), while the other group underwent LOD (n = 55). Rates of ovulation, pregnancy, and abortion were compared between both groups. RESULTS: Rates of normal menstruation, ovulation, and pregnancy in the LOD group were higher than in the Met group: 76.4% (42/55) vs. 58.2% (32/55), P < 0.04; 50.8% (11/258) vs. 33.5% (94/281), P < 0.001; 38.2% (21/55) vs. 20.0% (11/55), P < 0.03. The difference in the early abortion rate between both groups was not statistically significant. CONCLUSIONS: Although Met can significantly improve a patient's insulin resistance, we found that in patients diagnosed with CIRCPOS, LOD can be much more effective in improving rates of normal menstruation, ovulation, and pregnancy.

A unique mono- and diacylglycerol lipase from Penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity.

A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5-9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries.

Synthesis and biological evaluation of novel bis-aromatic amides as novel PTP1B inhibitors.

A series of bis-aromatic amides was designed, synthesized, and evaluated as a new class of inhibitors with IC50 values in the micromolar range against protein tyrosine phosphatase 1B (PTP1B). Among them, compound 15 displayed an IC50 value of 2.34±0.08 µM with 5-fold preference over TCPTP. More importantly, the treatment of CHO/HIR cells with compound 15 resulted in increased phosphorylation of insulin receptor (IR), which suggested extensive cellular activity of compound 15. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris.

BACKGROUND: Xylanases have attracted much attention because of their potential applications. Unfortunately, the commercialization of xylanases is limited by their low catalytic activities. The aim of this study was to improve the activity of a xylanase by optimization of the expression conditions and to investigate its characterization. RESULTS: The activity of recombinant AuXyn11A (reAuXyn11A), a family 11 xylanase from Aspergillus usamii E001 expressed in Pichia pastoris GS115, reached 912.6 U mL⁻¹ under the optimized conditions, which was 2.14 times as high as that expressed using the standard protocol. After the endogenous 18-aa propeptide had been processed in P. pastoris, reAuXyn11A (188-aa mature peptide) was secreted and purified with a specific activity of 22 714 U mg⁻¹. It displayed maximum activity at pH 5 and 50 °C and was stable in the pH range 4-8 and at a temperature of 45 °C or below. Its activity was not significantly affected by most metal ions and EDTA. Xylooligosaccharides ranging from xylobiose (X2) to xylohexaose (X6) were produced from insoluble corncob xylan by reAuXyn11A. CONCLUSION: Its high specific activity and good enzymatic properties suggest that reAuXyn11A is a potential candidate for applications in industrial processes.

Cloning, expression of a feruloyl esterase from Aspergillus usamii E001 and its applicability in generating ferulic acid from wheat bran.

A cDNA gene (AufaeA), which encodes a mature polypeptide of the type-A feruloyl esterase from Aspergillus usamii E001 (abbreviated to AuFaeA), was cloned and heterologously expressed in Pichia pastoris GS115. One transformant, labeled as P. pastoris GSFaeA4-8, expressing the highest recombinant AuFaeA (reAuFaeA) activity of 10.76 U/ml was selected by the flask expression test. The expressed reAuFaeA was purified to homogeneity with an apparent molecular weight of 36.0 kDa by SDS-PAGE analysis, and characterized using the model substrate of methyl ferulate (MFA). The purified reAuFaeA was optimally active at pH 5.0 and 45 °C, and highly stable at pH 4.0-6.5 and 45 °C or below. Its activity was not significantly affected by metal ions tested and EDTA. The K m and V max of reAuFaeA towards MFA were 4.64 mM and 115.5 U/mg, respectively. High-performance liquid chromatography analysis showed that only 9.7 % of total alkali-extractable ferulic acid (FA) was released from destarched wheat bran by reAuFaeA alone. The released FA increased to 36.5 % when reAuFaeA was used together with a recombinant Aspergillus usamii GH family 11 xylanase A, indicating a synergistic interaction between them.

Fusing a carbohydrate-binding module into the Aspergillus usamii ß-mannanase to improve its thermostability and cellulose-binding capacity by in silico design.

The AuMan5A, an acidophilic glycoside hydrolase (GH) family 5 ß-mannanase derived from Aspergillus usamii YL-01-78, consists of an only catalytic domain (CD). To perfect enzymatic properties of the AuMan5A, a family 1 carbohydrate-binding module (CBM) of the Trichoderma reesei cellobiohydrolase I (TrCBH I), having the lowest binding free energy with cellobiose, was selected by in silico design, and fused into its C-terminus forming a fusion ß-mannanase, designated as AuMan5A-CBM. Then, its encoding gene, Auman5A-cbm, was constructed as it was designed theoretically, and expressed in Pichia pastoris GS115. SDS-PAGE analysis displayed that both recombinant AuMan5A-CBM (reAuMan5A-CBM) and AuMan5A (reAuMan5A) were secreted into the cultured media with apparent molecular masses of 57.3 and 49.8 kDa, respectively. The temperature optimum of the reAuMan5A-CBM was 75°C, being 5°C higher than that of the reAuMan5A. They were stable at temperatures of 68 and 60°C, respectively. Compared with reAuMan5A, the reAuMan5A-CBM showed an obvious decrease in K m and a slight alteration in V max. In addition, the fusion of a CBM of the TrCBH I into the AuMan5A contributed to its cellulose-binding capacity.

Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation.

BACKGROUND: Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement. RESULTS: The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn1157, was predicted by molecular dynamics (MD) simulation. An NhXyn1157-encoding gene, Nhxyn1157, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn1157 (re-NhXyn1157) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn1157 were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²âº and Ag⁺. CONCLUSION: This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn1157 a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme.

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K(M) vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K(M)-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5-8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn(2+), which caused a strong inhibition. The K(m) and V(max), towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-ß-1,4-D-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.

Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution.

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS) . The suitable N-terminus of AoXyn11A to be replaced was selected by the comparison of B-factors between AoXyn11A and EvXyn11(TS) , which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N-terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half-life was 197-fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K(m) but a decrease in V(max).