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Laminaripentaose (L5)‐producing β‐1,3‐glucanases can preferentially cleave the triple‐helix curdlan into β‐1,3‐glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, β‐1,3‐glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a β‐1,3‐glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg−1). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/β) domain, which formed an unusually wide groove with a crescent‐like structure. In the two complex structures (SpGlu64A–L3 and SpGlu64A–L4), two oligosaccharide chains were captured and the triple‐helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple‐helical β‐1,3‐glucan. A catalytic framework (β6–β9–β10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype‐like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A–CBM) and a mutant (Y161F; by site‐directed mutation) were obtained, with 1.2‐ and 1.7‐fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A–CBM and ‐Y161F improved the enzyme activity by 2.63‐fold. The study will not only be helpful in understanding the reaction mechanism of β‐1,3‐glucanases but will also provide a basis for further enzyme engineering. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is one of the leading causes of cancer-related death in women. The early detection of breast cancer with screening mammograms plays a pivotal role in reducing mortality rates. Although population-based double-reading screening mammograms have reduced mortality by over 31% in women with breast cancer in Europe, continuing this program is difficult due to the shortage of radiologists. Artificial intelligence (AI) is an emerging technology that has provided promising results in medical imaging for disease detection. This study investigates the performance of AI models on an Australian mammographic database, demonstrating how transfer learning from a USA mammographic database to an Australian one, contrast enhancement on mammographic images and the quality of training data according to radiologists' concordance can improve breast cancer diagnosis. Our proposed methodology offers a more efficacious approach for AI to contribute to radiologists' decision making when interpreting mammography images. This paper investigates the adaptability of four state-of-the-art artificial intelligence (AI) models to the Australian mammographic context through transfer learning, explores the impact of image enhancement on model performance and analyses the relationship between AI outputs and histopathological features for clinical relevance and accuracy assessment. A total of 1712 screening mammograms (n = 856 cancer cases and n = 856 matched normal cases) were used in this study. The 856 cases with cancer lesions were annotated by two expert radiologists and the level of concordance between their annotations was used to establish two sets: a 'high-concordances subset' with 99% agreement of cancer location and an 'entire dataset' with all cases included. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of Globally aware Multiple Instance Classifier (GMIC), Global-Local Activation Maps (GLAM), I&H and End2End AI models, both in the pretrained and transfer learning modes, with and without applying the Contrast Limited Adaptive Histogram Equalization (CLAHE) algorithm. The four AI models with and without transfer learning in the high-concordance subset outperformed those in the entire dataset. Applying the CLAHE algorithm to mammograms improved the performance of the AI models. In the high-concordance subset with the transfer learning and CLAHE algorithm applied, the AUC of the GMIC model was highest (0.912), followed by the GLAM model (0.909), I&H (0.893) and End2End (0.875). There were significant differences (p < 0.05) in the performances of the four AI models between the high-concordance subset and the entire dataset. The AI models demonstrated significant differences in malignancy probability concerning different tumour size categories in mammograms. The performance of AI models was affected by several factors such as concordance classification, image enhancement and transfer learning. Mammograms with a strong concordance with radiologists' annotations, applying image enhancement and transfer learning could enhance the accuracy of AI models. [ABSTRACT FROM AUTHOR]

Objectives: This study explored the familiarity, perceptions and confidence of Australian radiology clinicians involved in reading screening mammograms, regarding artificial intelligence (AI) applications in breast cancer detection. Methods: Sixty-five radiologists, breast physicians and radiology trainees participated in an online survey that consisted of 23 multiple choice questions asking about their experience and familiarity with AI products. Furthermore, the survey asked about their confidence in using AI outputs and their preference for AI modes applied in a breast screening context. Participants' responses to questions were compared using Pearson's χ 2 test. Bonferroni-adjusted significance tests were used for pairwise comparisons. Results: Fifty-five percent of respondents had experience with AI in their workplaces, with automatic density measurement powered by machine learning being the most familiar AI product (69.4%). The top AI outputs with the highest ranks of perceived confidence were 'Displaying suspicious areas on mammograms with the percentage of cancer possibility' (67.8%) and 'Automatic mammogram classification (normal, benign, cancer, uncertain)' (64.6%). Radiology and breast physicians preferred using AI as second-reader mode (75.4% saying 'somewhat happy' to 'extremely happy') over triage (47.7%), pre-screening and first-reader modes (both with 26.2%) (P < 0.001). Conclusion: The majority of screen readers expressed increased confidence in utilising AI for highlighting suspicious areas on mammograms and for automatically classifying mammograms. They considered AI as an optimal second-reader mode being the most ideal use in a screening program. The findings provide valuable insights into the familiarities and expectations of radiologists and breast clinicians for the AI products that can enhance the effectiveness of the breast cancer screening programs, benefitting both healthcare professionals and patients alike. What is known about the topic? Artificial intelligence (AI) holds promise in providing computer-aided detection in health care, however, current research suggests that standalone AI applications in clinical practice fall short of matching the accuracy of a single radiologist. What does this paper add? The study showed a significant preference among clinicians for using AI as a supplementary tool, serving as a second-reader. Such an integrated approach, where AI aids in flagging suspicious areas on mammograms or offers automatic classification, reflects the ideal cooperation between breast screening readers and AI systems. What are the implications for practitioners? These insights shed light on clinicians' familiarity with and expectations of AI tools that can boost the effectiveness of breast screening programs. [ABSTRACT FROM AUTHOR]

This paper is a methodological reflection on a community-based participatory research (CBPR) project that used the photovoice method to unravel the educational experiences of disabled college students in China's higher education institutions. Although CBPR endeavors to nurture equitable community engagement in research processes, there are practical challenges to address the full participation of people with divergent disabilities and access needs. Drawing upon the critical disability studies literature, and particularly upon the activist scholarship of disability justice that revolves around interdependency, collective access, and cross-disability solidarity, we provide an open discussion on the complexities, tensions, and challenges of envisioning and creating access for participants with different disabilities in a visuality-centered CBPR. Our findings suggest that access creation in CBPR should go beyond the checklist style of accommodation and instead be approached as relational, dynamic, and iterative processes that require ongoing reflection, (re)learning, and negotiation among researchers and participants. We implemented nonvisual photography to adapt to the visuality-centered bias in the photovoice method. Nonvisual photography empowered participants with visual disabilities to evoke multiple sensorialities in their photo-taking, displaying, and interpretation. Moreover, the participants without visual disabilities also learned and practiced collective access and interdependency through co-transforming the CBPR into an accessible space for all. The heterogeneity among participants with divergent disabilities and access needs challenged CBPR's envisioning of a unified community with recognized commonalities. The inclusion of participants across disability groups created an opportunity for all participants to draw meaning from the ableist social and political circumstances that forged them as a community and to develop a sense of belonging and bonding in and through CBPR. In that light, disability justice and access should be reimagined and incorporated into CBPR. [ABSTRACT FROM AUTHOR]

Bio‒production of alginate oligosaccharides (AOSs), a type of functional food additive, is a promising way for green utilization of algae, in which alginate hydrolyzing enzymes play a key role. A novel alginate lyase gene (MiAly17A) from a marine bacterium Microbulbifer arenaceous was heterologously expressed in E. coli. The coding sequence of the gene shared the highest identity of 86% with a polysaccharide lyase (PL) family 17 alginate lyase (AlgL17) from Microbulbifer sp. ALW1. The recombinant enzyme (MiAly17A) was purified and biochemically characterized. MiAly17A showed maximal enzyme activity at 40 °C and pH 7.5, respectively. It was stable at the temperatures below 35 °C and within pH 5.0–8.0. The enzyme activities were increased by 5.3 and 5.6 folds in the presence of 100 mM of K+ and Na+, respectively. MiAly17A was bifunctional and could hydrolyze sodium alginate to release unsaturated monosaccharides and oligosaccharides with degrees of polymerization (DP) 2–7. The enzyme catalyzed the cleavage of glycosidic bonds from the non-reducing ends and the backbone of the tested oligosaccharides (DP ≥ 4), exhibiting both exolytic and endo-lytic activities. Moreover, MiAly17A was used for the production of alginate oligosaccharides from sodium alginate, and the highest conversion ratio of 68% was obtained. The unique properties may possess the enzyme great potential for preparation of alginate oligosaccharides. • A novel alginate lyase gene (MiAly17A) from Microbulbifer arenaceous was identified. • MiAly17A was heterologously expressed in E. coli and characterized. • MiAly17A was a salt-active enzyme, and was significantly activated by Na+ and K+. • MiAly17A exhibited both exolytic and endolytic cleavage activities. • MiAly17A was suitable for production of alginate oligosaccharides. [ABSTRACT FROM AUTHOR]

• A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was expressed in Pichia pastoris. • The highest β-1,3-1,4-glucanase activity of 159,500 U/mL was produced in 5-L fermentor. • AaBglu12A was an acid tolerant enzyme. • The enzyme exhibited strict substrate specificity towards various β-1,3-1,4-glucans. • AaBglu12A improved mashing performance of filtration rate and viscosity reduction. A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The K m values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The V max values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties. [ABSTRACT FROM AUTHOR]

A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200 U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 °C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 °C. Among tested p -nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p -nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4 U/mg and 1152.5 U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24 h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation. [ABSTRACT FROM AUTHOR]

A chitinase gene (RmChiA) encoding 445 amino acid (aa) residues from a fungus Rhizomucor miehei was cloned and overexpressed in Escherichia coli. Two kinds of RmChiA crystal forms, with space groups P3 2 2 1 and P1, were obtained by sitting-drop vapor diffusion and the structures were determined by X-ray diffraction. The overall structure of RmChiA monomer, which is the first structure of bacterial-type chitinases from nonpathogenic fungi, adopts a canonical triosephosphate isomerase (TIM) barrel fold with two protruding chitinase insertion domains. RmChiA exhibited a unique NxDxE catalytical motif and a real active site tunnel structure, which are firstly found in GH family 18 chitinases. The motif had high structural homolog with the typical DxDxE motif in other GH family 18 chitinases. The tunnel is formed by two unusual long loops, containing 15 aa and 45 aa respectively, linked by a disulfide bond across the substrate-binding cleft. Mutation experiments found that opening the roof of tunnel structure increased the hydrolysis efficiency of RmChiA, but the thermostability of the mutants decreased. Moreover, the tunnel structure endowed RmChiA with the exo -chitinase character. • RmChiA exhibited a unique NxDxE catalytical motif and a real active site tunnel structure. • The two unusual loops were locked by the disulfide bond to form a roof structure in the active tunnel. • Truncated mutants increased the hydrolysis efficiency, but decreased the thermostability. [ABSTRACT FROM AUTHOR]

Abstract: A novel thermophilic xylanase-producing fungus, Chaetomium sp. CQ31 produced 131Uml−1 of xylanase when grown on a medium containing corncob (3.5%, w/v) at 37°C for 6days. A low molecular xylanase was purified 6.5-fold to homogeneity with a recovery yield of 17.5%. Its molecular mass was estimated to be 25.1kDa by SDS–PAGE. The xylanase had an optimum pH of 7.5, and its optimal temperature was 65°C. Apparent K m values of the xylanase for birchwood, beechwood and oat-spelt xylan were 1.3, 0.86 and 4.4mg/ml, respectively. The influence of this xylanase on the quality of Chinese steamed bread (CSB) was further studied. Addition of xylanase in the range 2.5–5.0ppm caused a 20–24.5% increase in specific volume over the control and remarkable decrease (8.9–24.2%) in firmness was also noticed. This is the first report on the purification, characterisation and application of a xylanase from Chaetomium sp. CQ31. [Copyright &y& Elsevier]

Abstract: A cDNA library of Paecilomyces thermophila was constructed, and the gene encoding xylanase (designated Pt xynA) was isolated from the library. Pt xynA consisted of 681bp, and the translated protein encoded 226 amino acids. This is the first functional gene cloned from P. thermophila. The gene was successfully expressed in Escherichia coli BL21 and the recombinant xylanase (XynA) was purified to homogeneity by Ni–NTA and Sephadex G50. XynA showed an optimum activity at 75°C and pH 7.0. Its residual activity was more than 60% after being treated at 85°C for 30min. Km values of XynA for birchwood xylan, beechwood xylan and oat-spelt xylan were 4.4, 3.6 and 9.7mgml−1, respectively. The enzyme has an endohydrolytic mode of action and can hydrolyse xylotriose to xylobiose through transglycosylation. These results indicate the XynA is a thermostable enzyme and has great potential in various industries. [Copyright &y& Elsevier]

Abstract: A β-mannosidase gene (TM1624) from Thermotoga maritima MSB8, the hyperthermophilic bacterium was expressed as a soluble C-terminal His-tagged protein in E. coli. Heat treatment of cell lysate followed by metal affinity- and anion-exchange chromatographic techniques the recombinant β-mannosidase was purified to apparent homogeneity. The recovery of the purified protein from the crude lysate was 23%. Results of SDS-PAGE analysis (96.8kDa) and gel permeation chromatography (93.2kDa) indicated monomeric nature of the β-mannosidase protein. The enzyme displayed its maximal activity at pH 7.0 with pH stability over a range of pH 5.0–9.0. Similarly, the optimum temperature for maximal activity was found to be 95°C and thermostability of up to 85°C. The substrate specificity and kinetics of the enzyme was studied using different mannooligosaccharides and pNP-β-d-mannopyranoside. The K m value of the purified enzyme for pNPM was 0.49mM. Different mannooligosaccharides tested as enzyme substrates were hydrolysed in an exo-wise manner when checked by thin-layer chromatography (TLC). The enzyme also exhibited transglycosidase activity when the reaction was carried out with 10% (w/v) of mannobiose in the presence of alcohols or galactose. Because of extreme thermostability and transglyocosylation properties of β-mannosidase from T. maritima, the enzyme may be of industrial applications in future. This is the first report on the purification and characterization of a β-mannosidase from T. maritima. [Copyright &y& Elsevier]

Abstract: The effect of the recombinantly produced xylanase B (XynB) from Thermotoga maritima MSB8 on the quality of frozen partially baked bread (FPBB) was investigated. Addition of XynB to wheat flour dough resulted in a significant increase in dough extensibility (L), swelling (G), and a decrease in dough resistance to deformation (P), configuration. Bread crumb characteristics were studied by differential scanning calorimeter (DSC) and dynamic-mechanical analysis (DMA). The results show that addition of XynB leads to improvements in the bread quality of FPBB and retards bread staling compared to the control. The greatest improvements were obtained in specific volume (+35.2%) and crumb firmness (−40.0%). The control FPBB was significantly firmer in texture and higher in amylopectin recrystallization than the bread with XynB. During frozen storage of FPBB with and without XynB for 8 weeks, the crumb firmness increased gradually and the specific volume slightly decreased with the frozen storage time. The ΔH values of freezable water (FW) endothermic transitions increased with frozen storage time for all samples. However, addition of XynB lowered the ΔH values indicating a decrease in FW. Therefore, XynB is useful in improving the quality of FPBB. DMA was also used to monitor the shrinking behavior of the samples. Addition of XynB increased the contraction during chilling but significantly diminished the total shrinking and frozen-state shrinking of the bread crumb during the freezing process. [Copyright &y& Elsevier]

Abstract: A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8×107 Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel. [Copyright &y& Elsevier]

Abstract: A newly isolated Bacillus subtilis WY34 produced high level of mannanase (1105U/ml) when grown on konjac powder as the carbon source at 50°C. The β-mannanase was purified 5.4-fold to homogeneity with a final recovery of 20.3% and a specificity of 8302.4U/mg protein. The purified mannanase appeared as a single protein band on SDS–PAGE gel with a molecular mass of approx. 39.6kDa. It was identified as a glycoprotein by periodic acid-Schiff staining with a carbohydrate content of 13.1%. The first 10 N-terminal amino acid sequence (HTVSPVNPNA) of the purified enzyme showed high homology (90% identity) with the N-terminal region of β-mannanase from B. subtilis NM-39. The optimal temperature and pH for mannanase activity was 65°C and pH 6.0, respectively. The mannanase activity was stable within pH 5.5–10.1. It was stable up to 60°C at pH 6.0. The mannanase was highly specific towards locust bean gum, but exhibited very low activity towards starch, CMC and birchwood xylan. Apparent K m values of the mannanase for locust bean gum, guar gum and konjac powder were 7.6, 10.5 and 27.4mgml−1, respectively. Copra mannan was degraded mainly to mannotetraose, mannobiose and mannotriose when incubated with the mannanase, suggesting that the mannanase is an endomannanase and is suitable for mannooligosaccharide production. These unique properties of the purified β-mannanase from B. subtilis WY34 make this enzyme attractive for biotechnological applications. [Copyright &y& Elsevier]

Abstract: A novel lectin (AMML) was isolated from a Chinese herb, i.e., the roots of Astragalus mongholicus, using a combination of ammonium sulfate fraction and ion exchange chromatographies. The molecular mass of intact AMML was determined to be 66,396Da by MALDI-TOF mass spectrometry and 61.8kDa by gel filtration, respectively. AMML was a dimeric protein composed of two identical subunits each with a molecular mass of 29.6kDa. The lectin was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified lectin hemagglutinated both rabbit and human erythrocytes, and showed preference for blood types O (native) and AB (trypsin-treated). Among various carbohydrates tested, the lectin was best inhibited by d-galactose and its derivatives with pronounced preference for lactose (3.13mM). N-terminal amino acid sequence of AMML was determined as ESGINLQGDATLANN. The optimal pH range for lectin activity was between pH 4.5 and 7.5, and the lectin was active up to 65°C. It also exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola. [Copyright &y& Elsevier]

Abstract: A 47kDa xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was immobilized on Eudragit S-100 and an immobilization efficiency of 90% was obtained. The optimum pH of the immobilized xylanase shifted from 5.8 to 6.3 while its optimum temperature moved from 60 to 65°C. The immobilized enzyme also showed a higher thermal stability than the free xylanase. Only a marginal increase in the Km values of free enzyme was observed upon immobilization. However, Vmax values of the immobilized enzyme were 82–161% higher than those of the free xylanase. The immobilized xylanase was also evaluated for its application in hydrolyzing the corncob powder pre-treated with 2% NaOH solution for xylo-oligosaccharide production. The final extent of xylan hydrolysis using pre-treated corncob powder was 84% for the immobilized enzyme after 24h of incubation at 55°C. The immobilized xylanase retained 81% of its initial hydrolysis activity even after being recycled four times. Therefore, the immobilized xylanase is suitable to produce xylo-oligosaccahrides from corncob powder pre-treated with dilute alkaline solution. [Copyright &y& Elsevier]

The study was carried out to investigate the effect of hyperthermophilic xylanase B (XynB) from Thermotoga maritima on the properties of wheat bread and its staling during storage. The presence of XynB in the dough led to improvements in the breadmaking quality (i.e. specific volume and crumb structure) and provided an anti-staling effect on breads compared to the control. Addition of XynB could cause ≈60% increase in specific volume in comparison with the control. By fitting the crumb firming kinetics during storage to the Avrami equation, it showed that XynB retarded the bread staling by reducing the initial crumb firmness and the firming process during storage. XynB hydrolyzed the isolated WU-AX faster than WE-AX under the ratio of wheat AX in wheat flour. Improvement of bread quality by XynB can partly be ascribed to the enzyme specificity. [Copyright &y& Elsevier]

The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was characterized and was found to cleave p-nitrophenyl β-d-xyloside via the transglycosylation reaction in the previous study. XynB was activated in the presence of alcohols, and XynB activity was increased by iso-propanol (2 M) to 2.1-fold. This type of activation was investigated and was shown to be due to the transglycosylation activity with p-nitrophenyl β-d-xylobioside being converted to alkyl β-d-xylobiosides in the presence of XynB and alcohols. Through the transglycosylation reaction, alkyl β-xylosides and xylobiosides were simultaneously produced in the presence of xylan and alcohols. Primary alcohols were found to be the best acceptors. The highest yields of alkyl β-xylosides and xylobiosides were 33% and 50% of the total sugar, respectively. XynB showed a great ability to transfer xylose and xylobiose to secondary alcohol acceptors, and was unique for being able to synthesize the tertiary alkyl β-xylosides and xylobiosides with high yields of 18.2% and 11.6% of the total sugar, respectively. This is the first report of a xylanase with the ability to synthesize tertiary alkyl β-xylosides and xylobiosides. The specificity of the β-linkage was confirmed by the proton nuclear magnetic resonance (1H NMR). Thus, XynB of T. maritima appears to be an ideal enzyme for the synthesis of useful alkyl β-xylosides and xylobiosides. [Copyright &y& Elsevier]

Sodium alginate is one of the most abundant sustainable gum source for dietary fiber production. However, the preparation efficiencies of low viscosity soluble dietary fiber from sodium alginate remain low. Here, a novel alginate lyase gene (FsAly7) from Flammeovirga sp. was identified and high–level expressed in Pichia pastoris for low viscosity soluble dietary fiber production. The highest enzyme production of 3050 U mL−1 was achieved, which is by far the highest yield ever reported. FsAly7 was used for low viscosity soluble dietary fiber production from sodium alginate, and the highest degradation rate of 85.5 % was achieved under a high substrate content of 20 % (w / v). The molecular weight of obtained soluble dietary fiber converged to 10.75 kDa. FsAly7 catalyzed the cleavage of glycosidic bonds in alginate chains with formation of unsaturated non-reducing ends simultaneously in the degradation process, thus altered the chemical structures of hydrolysates. The soluble dietary fiber exhibited excellent properties, including low viscosity, high oil adsorption capacity activity (2.20 ± 0.03 g g−1) and high emulsifying activity (60.05 ± 2.96 mL/100 mL). This investigation may provide a novel alginate lyase catalyst as well as a solution for the efficient production of low viscosity soluble dietary fiber from sodium alginate. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Xylan is the major component of hemicelluloses, which are the second most abundant polysaccharides in nature, accounting for approximately one-third of all renewable organic carbon resources on earth. Efficient degradation of xylan is the prerequisite for biofuel production. Enzymatic degradation has been demonstrated to be more attractive due to low energy consumption and environmental friendliness, when compared with chemical degradation. Exo-xylanases, as a rate-limiting factor, play an important role in the xylose production. It is of great value to identify novel exo-xylanases for efficient bioconversion of xylan in biorefinery industry. Results: A novel glycoside hydrolase (GH) family 8 reducing-end xylose-releasing exo-oligoxylanase (Rex)-encoding gene (PbRex8) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The deduced amino acid sequence of PbRex8 shared the highest identity of 74% with a Rex from Bacillus halodurans. The recombinant enzyme (PbRex8) was purified and biochemically characterized. The optimal pH and temperature of PbRex8 were 5.5 and 55 °C, respectively. PbRex8 showed prominent activity on xylooligosaccharides (XOSs), and trace activity on xylan. It also exhibited β-1,3-1,4-glucanase and xylobiase activities. The enzyme efficiently converted corncob xylan to xylose coupled with a GH family 10 endo-xylanase, with a xylose yield of 83%. The crystal structure of PbRex8 was resolved at 1.88 Å. Structural comparison suggests that Arg67 can hydrogen-bond to xylose moieties in the -1 subsite, and Asn122 and Arg253 are close to xylose moieties in the -3 subsite, the hypotheses of which were further verified by mutation analysis. In addition, Trp205, Trp132, Tyr372, Tyr277 and Tyr369 in the grove of PbRex8 were found to involve in glucooligosaccharides interactions. This is the first report on a GH family 8 Rex from P. barengoltzii. Conclusions: A novel reducing-end xylose-releasing exo-oligoxylanase suitable for xylose production from corncobs was identified, biochemically characterized and structurally elucidated. The properties of PbRex8 may make it an excellent candidate in biorefinery industries. [ABSTRACT FROM AUTHOR]

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0–6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. Key points: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins. [ABSTRACT FROM AUTHOR]

A novel acidic exochitinase gene (Ptchi18a) from the thermophilic fungus Paecilomyces thermophila J18 was cloned and expressed in Pichia pastoris. The highest chitinase activity of 75.9 U/mL was obtained by high cell density fermentation, which represents the highest yield for fungal chitinases to date. The recombinant enzyme (Ptchi18a) exhibited maximal activity at pH 5.5 and 60 ℃, respectively. It hydrolyzed colloidal chitin to yield mainly N -acetyl chitobiose (93.6%, w/w), exhibiting a typical exo-type action manner. Ptchi18a efficiently degraded the cell walls of Ganoderma lucidum spores accompany with cellulose for the extraction of bioactive components. The extraction ratios of bioactive polysaccharides, lipids and triterpenoids reached to 3.18%, 16.37% and 1.89% (w/w), respectively, which were 27, 9 and 4 times higher than that without enzymatic pretreatment. The unique properties may make Ptchi18a a good candidate for the bioconversion of chitin materials as well as the bio-extraction of bioactive components from chitin-rich plants or microorganisms. [Display omitted] • A novel acidic exochitinase gene (Ptchi45) was expressed in Pichia pastoris. • The highest chitinase activity of 75.9 U/mL was obtained in a 5–L fermentor. • Ptchi45 was purified to homogeneity and biochemically characterized. • Ptchi45 is suitable for the production of high purity N -acetyl chitobiose. • Ptchi45 can be used to extract the bioactive components from G. lucidum spores. [ABSTRACT FROM AUTHOR]

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0–6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. Key points: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • Neoagarotetraose extended the lifespan by up to 33.3 % of natural aging mice. • Neoagarotetraose improved the cerebral neuronal injuries of natural aging mice. • Neoagarotetraose increased abundance of short-chain fatty acids-producing bacteria. • Neoagarotetraose regulated the glucolipid and bile acid metabolism-related metabolic pathways. • Neoagarotetraose ameliorated cognitive impairment of Alzheimer's disease mice. Dietary oligosaccharides can impact the gut microbiota and confer tremendous health benefits. The aim of this study was to determine the impact of a novel functional oligosaccharide, neoagarotetraose (NAT), on aging in mice. 8-month-old C57BL/6J mice as the natural aging mice model were orally administered with NAT for 12 months. The preventive effect of NAT in Alzheimer's disease (AD) mice was further evaluated. Aging related indicators, neuropathology, gut microbiota and short-chain fatty acids (SCFAs) in cecal contents were analyzed. NAT treatment extended the lifespan of these mice by up to 33.3 %. Furthermore, these mice showed the improved aging characteristics and decreased injuries in cerebral neurons. Dietary NAT significantly delayed DNA damage in the brain, and inhibited reduction of tight junction protein in the colon. A significant increase at gut bacterial genus level (such as Lactobacillus , Butyricimonas , and Akkermansia) accompanied by increasing concentrations of SCFAs in cecal contents was observed after NAT treatment. Functional profiling of gut microbiota composition indicated that NAT treatment regulated the glucolipid and bile acid-related metabolic pathways. Interestingly, NAT treatment ameliorated cognitive impairment, attenuated amyloid-β (Aβ) and Tau pathology, and regulated the gut microbiota composition and SCFAs receptor-related pathway of Alzheimer's disease (AD) mice. NAT mitigated age-associated cerebral injury in mice through gut-brain axis. The findings provide novel evidence for the effect of NAT on anti-aging, and highlight the potential application of NAT as an effective intervention against age-related diseases. [ABSTRACT FROM AUTHOR]

Lacto- N -tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. β-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of β-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical characterization of a novel β-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest identity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55°C. It was stable within the pH range of 3.5 to 7.0 and up to 60°C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the galactose residue from o -nitrophenyl-β-galactopyranoside to lacto- N -triose II. Under optimal conditions, the conversion rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a β-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis. [ABSTRACT FROM AUTHOR]

Milk protein hydrolysate was optimally prepared by Protamex and PaproA (MP‐PP) exhibiting excellent angiotensin I‐converting enzyme (ACE) inhibitory activity (89.6%) at 0.5 mg/mL and protein recovery rate (79.0%). Meanwhile, MP‐PP was stable for acid–base and heat treatments, and even presented 80.5% of ACE inhibitory activity after handling in gastrointestinal fluids. However, transepithelial transportation via Caco‐2 cell monolayer lowered ACE inhibition of MP‐PP. Following the fractionation of MP‐PP, IESPPEI was identified as an outstanding ACE inhibitory peptide (IC50 of 6.4 μM), comparable with commercial VPP and IPP. Overall, MP‐PP and IESPPEI are potential functional ingredients to develop antihypertensive products. [ABSTRACT FROM AUTHOR]

Abstract Fermentation conditions for β-1,3-1,4-glucanase (TaGlu34) production in submerged culture by a thermophilic fungus, Thermoascus aurantiacus CAU830 were optimized. The highest enzyme activity of 3741 U/mL was obtained, and the crude enzyme was purified to homogeneity with a purification fold of 7.3 and a recovery yield of 11.6%. The molecular mass of the purified enzyme was estimated to be approximately 34 kDa on SDS-PAGE. TaGlu34 was most active at pH 6.0 and 75 °C, respectively. It showed excellent thermostability with thermal denaturing half-lives of 209, 130 and 69 min at 50, 60 and 70 °C, respectively. TaGlu34 exhibited strict substrate specificity towards barley β-glucan (13,527 U/mg), oat β-glucan (12,502 U/mg) and lichenan (9225 U/mg), but displayed no activity on other tested polysaccharides including laminarin, xylan, pullulan, CMC and starch. TaGlu34 hydrolyzed barley β-glucan and lichenan to yield both mainly disaccharide and trisaccharide, suggesting that it should be an endo type β-1,3-1,4-glucanase. Furthermore, TaGlu34 efficiently degraded the β-glucan component in oat bran to produce mainly oligosaccharides with degrees of polymerization (DP) 3–5, with the highest conversion ratio of 47.1%. The high yield and excellent enzymatic properties of TaGlu34 may make it a good candidate in industries. Graphical abstract Image 1 Highlights • A thermostable β-1,3-1,4-glucanase (TaGlu34) was purified and characterized. • TaGlu34 was most active at pH 6.0 and 75 °C and stable up to 70 °C. • TaGlu34 exhibited strict specificity on barley β-glucan, oat β-glucan and lichenan. • TaGlu34 degraded oat bran β-glucan to produce oligosaccharides with yield of 47.1%. [ABSTRACT FROM AUTHOR]

A novel xylanase gene ( PbXyn10A ) from Paenibacillus barengoltzii was cloned and expressed in Escherichia coli . PbXyn10A had an open reading frame of 3,063 bp, and its deduced amino acid sequence shared the highest identity of 72% with a xylanase from Paenibacillus curdlanolyticus. The recombinant xylanase ( Pb Xyn10A) was purified and biochemically characterized. Pb Xyn10A was most active at pH 6.5 and 60 °C, respectively. It exhibited strict substrate specificity towards birchwood xylan, beechwood xylan and oat-spelt xylan, with K m values of 2.19, 2.04 and 2.51 mg/mL, respectively. The enzyme hydrolyzed xylan to yield mainly xylooligosaccharides (XOS) with degree of polymerization 2–4. A new strategy for XOS production from corncobs pretreated by steam explosion using acidic electrolyzed water, followed by enzymatic hydrolysis was developed. The highest XOS yield of 75% (based on xylan in raw corncobs) was achieved. This is the first report on a xylanase from Paenibacillus barengoltzii . [ABSTRACT FROM AUTHOR]

A novel β-1,3-glucanase gene ( PaBglu50A ) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli . The deduced amino acid sequence of PaBglu50A showed the highest identity of 34% with the β-agarase belonging to glycoside hydrolase (GH) family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45 °C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40 °C. The K m and V max of PaBglu50A for curdlan and laminarin were 94.4 mg ml −1 and 23.4 μmol min −1 mg −1 , 3.65 mg ml −1 and 8.89 μmol min −1 mg −1 , respectively. All characterized members of GH family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo -β-1,3-glucanase, making a significant difference from the reported enzymes of GH family 50. These results suggested that PaBglu50A is the first endo -type β-1,3-glucanase (EC 3.2.1.39) in GH family 50. [ABSTRACT FROM AUTHOR]

A β-galactosidase gene ( PbBGal2A ) was cloned from Paenibacillus barengoltzii and expressed in Escherichia coli . The in silico analysis of the deduced amino acid sequences revealed that PbBGal2A shared the highest identity of 40% with the characterized glycoside hydrolase (GH) family 2 β-galactosidase from Actinobacillus pleuropneumoniae . The recombinant β-galactosidase (PbBGal2A) was purified with a molecular mass of 124.2 kDa on SDS-PAGE. The optimal pH and temperature of PbBGal2A were determined to be pH 7.5 and 45 °C, respectively. PbBGal2A was stable within pH 6.0-8.0 and up to 45 °C. It completely hydrolyzed the lactose in milk and whey powder solution. In addition, PbBGal2A exhibited high transglycosylation activity and a maximum yield of 47.9% (w/w) for galactooligosaccharides (GOS) production was obtained in 8 h at a lactose concentration of 350 g/L. These properties make PbBGal2A an ideal candidate for commercial use in the production of lactose-free milk and GOS. [ABSTRACT FROM AUTHOR]

Visual tracking of cells has many challenges due to complex situations such as varying cell population densities, intricate motion patterns and sophisticated interactions with other cells. This paper focuses on an efficient and effective ant-based method with working modes updated to track multiple cells over varying densities in the presence of occlusion or clustering situations. To overcome these challenges, the proposed Ant Colony Optimization (ACO) algorithm models two types of ant working modes, namely, cooperation mode and interactive competition mode, whereas the classical ACO algorithms use only one type of mode which performs poor in clutter situations. Moreover, mode update strategies based on ant colony pheromone is used to adjust pheromone field to obtain accurate state vector of cells. Experimental results demonstrate that the proposed method robustly tracks multiple cells in various scenarios. The averaged LSR, LTR and FTR of our method can be only 1.43, 1.71 and 1.37 %, respectively. Our experimental results also show that our tracking method is competitive with state-of-the-art multi-cell tracking methods. [ABSTRACT FROM AUTHOR]

A novel xylanase gene (CsXyn11A) from Chaetomium sp. CQ31 was extracellularly expressed in Aspergillus niger. CsXyn11A belonging to glycoside hydrolase (GH) family 11 shared 72% amino acid identity with the xylanase from Achaetomium sp. Xz-8. High xylanase activity of 3824 U/mL with 10.4 g/L protein concentration was secreted by recombinant A. niger in a 5-L fermentor. CsXyn11A was purified by Q-Sepharose ion-exchange chromatography column. It was optimally active at 70 °C and pH 7.0, respectively. The xylanase displayed strict substrate specificity towards different xylans, and showed high activity (1850 U/mg) towards wheat arabinoxylan. This enzyme was further applied in bread making to evaluate its effect on the bread quality. The incorporation of CsXyn11A reduced the development time, stable time and different torque values of dough samples. Addition of CsXyn11A (5 ppm) into the bread making remarkably improved the specific volume by 25.6% and decreased the hardness by 46%. The favorable properties of CsXyn11A suggest that it may be a promise candidate suitable for bread making. [Display omitted] • A novel xylanase from Chaetomium sp. (CsXyn11A) expressed in A. niger secreted 3824 U/mL. • CsXyn11A was optimally active at 70 °C and pH 7.0, respectively. • CsXyn11A displayed high specific activity of 1850 U/mg towards wheat arabinoxylan. • CsXyn11A significantly improved the bread specific volume (25.6%) and decreased crumb hardness (46%). [ABSTRACT FROM AUTHOR]

Prolyl oligopeptidases (POPs) are atypical serine proteases that are unique in their involvement in the maturation and degradation of prolyl‐containing peptide hormones and neuropeptides. They are potential pharmaceutical targets for the treatment of several neurodegenerative disorders, such as Alzheimer's disease. In this study, the catalytic and substrate‐regulatory mechanisms of a novel bacterial POP from Microbulbifer arenaceous (MaPOP) were investigated. The crystal structure revealed that the catalytic triad of MaPOP was covered by the central tunnel of an unusual β‐propeller domain. The tunnel not only provided the sole access to the active site for oligopeptides, but also protected large structured peptides or proteins from accidental proteolysis. The enzyme was able to cleave angiotensin I specifically at the carboxyl side of the internal proline residue, but could not hydrolyze long‐chain bovine insulin B in vitro. Like the ligand‐free structure, MaPOP bound to the transition‐state analog inhibitor ZPR was also in a closed state, which was not modulated by the common 'latching loop' found in other POPs. The substrate‐assisted catalytic mechanism of MaPOP reported here may represent a common mechanism for all POPs. These results may facilitate a better understanding of the catalytic behavior of POPs under physiological conditions. [ABSTRACT FROM AUTHOR]

The second α-galactosidase gene (designated as RmgalB ) was cloned from the thermophilic fungus Rhizomucor miehei and expressed in Pichia pastoris . The gene belonging to glycoside hydrolase (GH) family 36 has an open reading frame (ORF) of 2241 bp encoding 746 amino acids with two introns. The recombinant α-galactosidase (RmgalB) was secreted at high levels of 1953.9 U ml −1 in high cell density fermentor, which is the highest yield obtained for a α-galactosidase. The purified enzyme as a tetramer gave a single band corresponding to a molecular mass of 83.1 kDa in SDS–PAGE. The enzyme exhibited a very high specific activity of 505.5 U mg −1 . The optimum temperature and pH of RmgalB were determined to be 55 °C and pH 5.5, respectively. It was stable within pH 5.5–9.5 and up to 55 °C. RmgalB displayed specificity toward raffinose and stachyose, and completely hydrolyzed the anti-nutritive raffinose family oligosaccharides (RFOs). These properties make RmgalB useful in the food and feed industries. [ABSTRACT FROM AUTHOR]

[Display omitted] • A chitinase (PbChi70) from P. barengoltzii was engineered by directed evolution. • The specific activity of engineered enzyme (mPbChi70) was improved by 2.4 folds. • A two-enzyme system consisting of mPbChi70 and GlcNAcase was created in A. niger. • The cocktail showed high hydrolysis efficiency with chitin conversion rate of 71.9 %. • The purity of GlcNAc was up to 95 % (w/w) without additional purification processes. A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously β- N -acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials. [ABSTRACT FROM AUTHOR]

Soybeans are usually fermented by Bacillus subtilis to produce shuidouchi, which is a traditional fermentation soybean product in China. In the study, green soybeans were fermented by Bacillus velezensis to make a novel green soybean shuidouchi with multibioactivities. The processing conditions were optimized as follows: initial moisture content 75%, inoculum concentration 7 log CFU/g, and incubation time 24 h for prefermentation; water addition 50%, salt addition 6%, temperature 45°C, 3 days for postfermentation. The fermented green soybean shuidouchi (FGSS) showed 234.8 FU/g dry weight (DW) for the fibrinolytic activity and IC50 of 0.33 mg/ml for the anticoagulant activity. FGSS had higher contents of chemical components including 3.6 mg rutin (RE)/g DW of total flavonoids, 8.2 mg gallic acid (GAE)/g DW of total phenolics, 63.7 mg/g DW of reducing sugars, and 163.8 mg/g DW of peptides than the unfermented green soybean shuidouchi (UGSS). Moreover, it exhibited high antioxidant activities of 29.8, 85.1 μmol trolox equivalent (TE)/g DW, and 12.8 μmol Fe2+/g DW through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2'‐azino‐bis (3‐ethylbenzthiazoline‐6‐sulfonic acid) diammonium salt (ABTS), and ferric reducing antioxidant power (FRAP) experiments. Thus, a novel green soybean shuidouchi fermented by B. velezensis owing to multibioactivities can provide a theoretical basis for the further development of functional shuidouchi. [ABSTRACT FROM AUTHOR]

Abstract: The production of xylooligosaccharides (XOs) from corncobs was studied using a two-stage process based on a steam explosion pretreatment followed by enzymatic hydrolysis. Corncobs with different chip sizes were subjected to steam explosion under different experimental conditions of temperature and time, namely 188–204°C for 2.5–7.5min. The results indicate that corncobs were optimally steam exploded at 196°C for 5min, resulting in hemicellulose recovery of 22.8%. Especially, corncobs with large chip sizes (∼100mm) during steam explosion pretreatment were suitable to produce XOs. Furthermore, a thermostable xylanase from Paecilomyces themophila J18 was used to hydrolyze steam explosion liquor of corncobs (SELC) for the production of XOs. A maximum XOs yield of 28.6g XOs/100g xylan in corncobs was achieved and XOs syrup contained more than 90% of xylobiose and xylotriose when the hydrolysis was carried out under the optimized conditions (pH 7.0, 70°C, 7.5UmL−1 and 2.5h). These results suggest that the process might be effective in production of XOs for industrial applications. [Copyright &y& Elsevier]

In this study, a novel β-1,3-1,4-glucanase gene (designated as PtLic16A) from Paecilomyces thermophila was cloned and sequenced. PtLic16A has an open reading frame of 945 bp, encoding 314 amino acids. The deduced amino acid sequence shares the highest identity (61%) with the putative endo-1,3(4)-β-glucanase from Neosartorya fischeri NRRL 181. PtLic16A was cloned into a vector pPIC9K and was expressed successfully in Pichia pastoris as active extracellular β-1,3-1,4-glucanase. The recombinant β-1,3-1,4-glucanase (PtLic16A) was secreted predominantly into the medium which comprised up to 85% of the total extracellular proteins and reached a protein concentration of 9.1 g l with an activity of 55,300 U ml in 5-l fermentor culture. The enzyme was then purified using two steps, ion exchange chromatography, and gel filtration chromatography. The purified enzyme had a molecular mass of 38.5 kDa on SDS-PAGE. It was optimally active at pH 7.0 and a temperature of 70°C. Furthermore, the enzyme exhibited strict specificity for β-1,3-1,4- d-glucans. This is the first report on the cloning and expression of a β-1,3-1,4-glucanase gene from Paecilomyces sp. [ABSTRACT FROM AUTHOR]

Abstract: An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2kDa. However, it had a very low apparent molecular mass of 3.3kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65°C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55°C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis. [Copyright &y& Elsevier]

Abstract: A β-galactosidase gene (TM_0310) of Thermotoga maritima MSB8 was expressed in Escherichia coli. The recombinant β-galactosidase (designated BgalB) was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. BgalB belongs to the glycoside hydrolase family 42. Its molecular mass was estimated to be 78kDa and 76kDa by SDS–PAGE and gel filtration, respectively. The enzyme was optimum at pH 5.5, and it was quite stable over the pH range 5.0–11.4 at 70°C. It was optimally active at 80°C and was stable up to 75°C. Besides, BgalB exhibited broad substrate specificity with a preference for p-nitrophenyl-β-galactopyranoside (pNPGal). K m values of the purified enzyme for pNPGal, o-nitrophenyl-β-galactopyranoside (oNPGal) and pNP-β-fucopyranoside were 2.7mM, 12.5mM and 1.4mM, respectively. These properties make this enzyme an interesting candidate for biotechnological applications. This is the first report of the family 42 β-galactosidases from T. maritima. [Copyright &y& Elsevier]

Abstract: In the present study, an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and ammonium sulphate ((NH4)2SO4) was used to purify a thermostable xylanase produced by the thermophilic fungus Paecilomyces thermophila J18 in solid-state fermentation (SSF). The effects of PEG, (NH4)2SO4, loading mass and pH on xylanase partition in ATPS were investigated. Xylanase partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. The ATPS composition (w/w) of 12.5% PEG-4000, 25% (NH4)2SO4 and 50% enzyme solution at pH 7.2 was proved to be an excellent system for purification of xylanase in a single-step operation. SDS-PAGE analysis revealed that the purified xylanase was near homogeneity. A purification factor of 5.54 and a 98.7% enzyme yield were achieved in the top phase of this system. However, the xylanase produced in SSF was purified to homogeneity with a recovery yield of 34.5% by three steps. Therefore, ATPS provides an efficient and attractive method for purifying the xylanase in SSF. [Copyright &y& Elsevier]

Abstract: A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60°C, respectively. The RNase activity was stable within pH 5.0–11.0. It was stable up to 60°C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins. [Copyright &y& Elsevier]

• An alkaline phospholipase C gene (AoPC) from Aspergillus oryzae was cloned. • The PLC gene was co-expressed in P. pastoris with molecular chaperone PDI. • The PLC yield of 82, 782 U mL−1 was obtained in a 5-L fermentor. • AoPC was a cold-active enzyme having an optimal temperature of 25 °C. • AoPC significantly reduced the phosphorus of crude soybean oil by 93.3%. A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL−1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5–9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg−1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry. [ABSTRACT FROM AUTHOR]

A chitinase gene (PxChi52) from Paenibacillus xylanexedens Z2–4 was cloned and heterologously expressed in Escherichia coli BL21 (DE3). PxChi52 shared the highest identity of 91% with a glycoside hydrolase family 18 chitinase (ChiD) from Bacillus circulans. The recombinant enzyme (PxChi52) was purified and biochemically characterized. PxChi52 had a molecular mass of 52.8 kDa. It was most active at pH 4.5 and 65 °C, respectively, and stable in a wide pH range of 4.0–13.0 and up to 50 °C. The enzyme exhibited the highest specific activity of 16.0 U/mg towards colloidal chitin, followed by ethylene glycol chitin (5.4 U/mg) and ball milled chitin (0.4 U/mg). The K m and V max values of PxChi52 towards colloidal chitin were determined to be 3.06 mg/mL and 71.38 U/mg, respectively, PxChi52 hydrolyzed colloidal chitin and chitooligosaccharides with degree of polymerization 2–5 to release mainly N -acetyl chitobiose. In addition, PxChi52 displayed inhibition effects on the growth of some phytopathogenic fungi, including Alternaria alstroemeriae , Botrytis cinerea , Rhizoctonia solani , Sclerotinia sclerotiorum and Valsa mali. The unique properties of PxChi52 may enable it potential application in agriculture field as a biocontrol agent. [ABSTRACT FROM AUTHOR]

Synbiotic dietary supplements, as an effective means of regulating the gut microbiota, may have a beneficial effect on constipation. This study evaluated the effects of synbiotic yogurt containing konjac mannan oligosaccharides (KMOS) and Bifidobacterium animalis ssp. lactis BB12 (BB12) on constipated Kunming mice (the model group). Following administration of yogurt containing 2.0% KMOS and BB12 (YBK2.0), black fecal weight and number and gastrointestinal transit rate increased by 97.5, 106.3, and 55.7%, respectively, compared with the model group. Serum levels of excitability neurotransmitters (motilin, substance P, and acetylcholine) in the YBK2.0 group were increased by 139.7, 120.4, and 91.8%, respectively, and serum levels of inhibitory neurotransmitters (vasoactive intestinal peptide, nitric oxide, and acetylcholine) were decreased. Moreover, synbiotic yogurt supplementation significantly downregulated the expression of vasoactive intestinal peptide receptor 1 (VIPR1) and upregulated the expression of serotonin receptor 4 (5-HT4) in the colon, and enhanced the expression of the stem cell factor (SCF)/c-Kit pathway. Additionally, YBK2.0 treatment significantly regulated the community composition and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of gut microbiota, which were positively correlated with physiological parameters of constipation. Thus, supplementation with synbiotic yogurt composed of KMOS and BB12 could facilitate fecal excretion by regulating related pathways and the gut microbiota. These findings demonstrated that the synbiotic yogurt can be considered a functional food for alleviating constipation. [ABSTRACT FROM AUTHOR]

• A novel β- N -acetylhexosaminidase gene (CmHex187) was expressed in E. coli. • CmHex187 was purified and biochemically characterized. • An efficient bioprocess for LNT2 synthesis was developed. • The LNT2 conversion ratio of 44.3 % was obtained. • LNT2 exhibited higher prebiotic activity compared to fructooligosaccharides (FOS). β- N -acetylhexosaminidases have gained much attention for synthesis of functional oligosaccharides, but the relatively low conversion ratio limited their industrial applications. A β- N -acetylhexosaminidase gene (CmHex187) from Catenibacterium mitsuokai was successfully expressed in Escherichia coli. The recombinant enzyme (CmHex187) was purified, biochemically characterized and subjected to the synthesis of human milk oligosaccharides (HMOs). CmHex187 displayed optimal pH and temperature of 5.5 and 45 °C, respectively. The enzyme showed strong transglycosylation activity, as it efficiently catalyzed the synthesis of an important component of HMO lacto- N -triose II (LNT2) using 0.015 M p -nitrophenyl N -acetyl-β- d -glucosaminide and 1.2 M lactose at pH 7.0 and 60 °C for 1.5 h, with a high conversion ratio of 44.3 %. The formed LNT2 promoted the growth of tested Lactobacillus and Bifidobacteria strains. The excellent enzymatic properties and high LNT2 synthesis ability of CmHex187 may make it a good candidate in LNT2 production. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are important in food fermentation and may enhance overall host health. Previous studies to explore LAB metabolism mainly focused on the genera Lacticaseibacillus and Lactococcus. Pediococcus pentosaceus, historically recognized as an important food fermentation bacterial strain, can produce bacteriocins and occasionally demonstrated probiotic functionalities. This study thoroughly surveyed the growth kinetic of three P. pentosaceus isolates in various culture formulations, especially in fructooligosaccharide (FOS), xylooligosaccharide (XOS), or konjac mannooligosaccharide (KMOS) conditions. Results showed that P. pentosaceus effectively metabolized KMOS, the culture of which led to 23.6-fold population increase. However, FOS and XOS were less metabolized by P. pentosaceus. On functional oligosaccharide cultures, P. pentosaceus could result in higher population proliferation, more acidified fermentation environment, and higher glycoside hydrolysis activities in the culture. RNA-Seq analysis classified 1572 out of 1708 putative genes as mRNA-coding genes. The dataset also revealed that the three functional oligosaccharides led to extensive global functional gene regulations. Phosphate conservation and utilization efficiency enhancement may serve as a leading transcriptional regulation direction in functional oligosaccharide metabolisms. In summary, these discovered metabolic characteristics could be employed to support future studies. Key points: • Konjac mannooligosaccharides effectively promoted P. pentosaceus proliferation. • Functional genes were highly regulated in functional oligosaccharide utilization. • Phosphate conservation was an important transcriptional regulation direction. [ABSTRACT FROM AUTHOR]

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon–optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz–8. CsXynBop was over–expressed in Pichia pastoris GS115 by high–cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0–9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat–spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2–5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry. [ABSTRACT FROM AUTHOR]

A novel phospholipase C gene (TtPLC) from Thielavia terrestris CAU709 was cloned and efficiently expressed in Pichia pastoris. The deduced protein sequence of TtPLC shared the highest identity of 33% with the characterized phospholipase C from Arabidopsis thaliana. The highest phospholipase C yield of 98, 970 U mL−1, with a protein concentration of 4.9 mg mL−1 was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant enzyme (TtPLC) was purified to homogeneity with a recovery yield of 59.1% and a specific activity of 22, 910 U mg−1. TtPLC was most active at pH 6.5 and 55 °C, respectively. It was stable within the pH range of 4.5–8.0 and up to 45 °C. The enzyme exhibited excellent stability in different surfactants and organic solvents, including Tween 20 (147.6%), Tween 40 (180.6%), Tween 60 (205.4%), cyclohexane (160.0%), n-octane (178.2%), n-heptane (180.7%), acetone (187.5%) etc. The application of TtPLC in crude soybean oil degumming process significantly reduced the residual phosphorus content from 135.4 mg kg−1 to 7.9 mg kg−1 under the optimized conditions, which satisfied the requirement of environmental friendly physical refining process for oil refining industry. Therefore, TtPLC should be a good candidate in oil refining industry. • A novel phospholipase C gene (TtPLC) from Thielavia terrestris was cloned. • TtPLC was expressed in Pichia pastoris with the highest yield of 98,970 U mL−1. • The recombinant enzyme (TtPLC) was purified and biochemically characterized. • TtPLC was most active at pH 6.5 and 55 °C. • TtPLC showed application potential in the degumming of crude soybean oil. [ABSTRACT FROM AUTHOR]

• The α-galactosidase gene (PtGal36A) from Paecilomyces thermophila was expressed in E. coli. • The recombinant enzyme (PtGal36A) was optimally active at pH 5.0 and 60 °C, respectively. • PtGal36A efficiently degraded raffinose family oligosaccharides (RFOs) in soybean meal. • PtGal36A exhibited excellent stability in the presence of various proteases. • PtGal36A may be a good candidate for potential applications in food and feed industries. A novel glycoside hydrolase (GH) family 36 α-galactosidase gene (designated PtGal36A) from Paecilomyces thermophila was cloned and expressed in Escherichia coli. The deduced sequence of the gene shared the highest identity of 87% with the characterized α-galactosidase from Aspergillus nidulans FGSC A4. The recombinant enzyme (PtGal36A) was purified to homogeneity with a purification fold of 11.0 and a recovery yield of 55.2%. PtGal36A was most active at pH 5.0 and 60 °C and was stable within the pH range of 4.5-11.5 and up to 50 °C. PtGal36A displayed strict specific activity towards substrates with α-galactosyl linkages in the nonreducing ends, with the highest activity on stachyose (58.5 U/mg), followed by melibiose (39.2 U/mg) and raffinose (31.4 U/mg). The enzyme efficiently hydrolyzed raffinose family oligosaccharides in soybean meal by more than 95%. Moreover, PtGal36A showed excellent resistance (residual activities >90%) against α-chymotrypsin, proteinase K, subtilisin A, trypsin and papain. Therefore, PtGal36A should be a good candidate for the food and feed industries. [ABSTRACT FROM AUTHOR]