Abstract:Xylan is the most abundant hemicellulosic component, and its degradation is the major step in a wide variety of industrial processes. Xylanases, which are capable of randomly hydrolyzing the β‐1,4‐xylosidic linkages, constitute the key enzymes in xylan decomposition. As xylanases hold a high economic potential in the marketplace, great efforts have been made in search of suitable candidates for the development into commercial products with better performances such as higher specific activity, better thermostab… Show more
“…Moreover, the β-turn linked with 207-SSGS-210 in NFX is more compact than that in XynCDBFV. It is well known that the GH11 xylanase generally folds into a β-jelly-roll structure [8], so the stability of GH11 xylanase is highly related with the arrangement and compactness of β-sheets. Analogous residues 207–210 with different secondary structures may contribute to various stabilities among GH11xylanases.Fig.…”
BackgroundXylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus Neocallimastix patriciarum, by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment.ResultsC-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (K
m, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (k
cat
/K
m) of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability.ConclusionsThis is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0824-y) contains supplementary material, which is available to authorized users.
“…Moreover, the β-turn linked with 207-SSGS-210 in NFX is more compact than that in XynCDBFV. It is well known that the GH11 xylanase generally folds into a β-jelly-roll structure [8], so the stability of GH11 xylanase is highly related with the arrangement and compactness of β-sheets. Analogous residues 207–210 with different secondary structures may contribute to various stabilities among GH11xylanases.Fig.…”
BackgroundXylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus Neocallimastix patriciarum, by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment.ResultsC-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (K
m, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (k
cat
/K
m) of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability.ConclusionsThis is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0824-y) contains supplementary material, which is available to authorized users.
“…The presence of extra disulfide and salt bridges, hydrophobic side chains, and N-terminal proline residues helps in reduction of conformational freedom of the protein structure. Thus, it help in providing more stability to protein at the higher temperature (Turunen et al 2001;Chen et al 2015). Different structural modifications such as high Thr/Ser ratio and high charged residues, i.e., Arg, cause enhanced polar interaction and improved stabilization of the alphahelix region and secondary structures (Hakulinen et al 2003).…”
Section: Structural Properties Of Xylanase Responsible For Thermal Anmentioning
confidence: 99%
“…Different structural modifications such as high Thr/Ser ratio and high charged residues, i.e., Arg, cause enhanced polar interaction and improved stabilization of the alphahelix region and secondary structures (Hakulinen et al 2003). The xylanase protein has a large number of ion pairs/aromatic residues on the surface of protein resulting in enhanced interactions (Polizeli et al 2005;Chen et al 2015). The low average protein rigidity i.e.…”
Section: Structural Properties Of Xylanase Responsible For Thermal Anmentioning
Xylan is the second most abundant naturally occurring renewable polysaccharide available on earth. It is a complex heteropolysaccharide consisting of different monosaccharides such as l-arabinose, d-galactose, d-mannoses and organic acids such as acetic acid, ferulic acid, glucuronic acid interwoven together with help of glycosidic and ester bonds. The breakdown of xylan is restricted due to its heterogeneous nature and it can be overcome by xylanases which are capable of cleaving the heterogeneous β-1,4-glycoside linkage. Xylanases are abundantly present in nature (e.g., molluscs, insects and microorganisms) and several microorganisms such as bacteria, fungi, yeast, and algae are used extensively for its production. Microbial xylanases show varying substrate specificities and biochemical properties which makes it suitable for various applications in industrial and biotechnological sectors. The suitability of xylanases for its application in food and feed, paper and pulp, textile, pharmaceuticals, and lignocellulosic biorefinery has led to an increase in demand of xylanases globally. The present review gives an insight of using microbial xylanases as an "Emerging Green Tool" along with its current status and future prospective.
“…Di sisi lain, terdapat enzim hidrolitik mananase (EC 3.2.1.x) yang secara acak memotong ikatan 1,4 pada rantai utama gl ukomanan, galaktomanan, dan galaktoglukomanan (Collins, Gerday & Feller, 2005;McCleary, 1988). Enzim xilanase dan mananase banyak diaplikasikan dalam kegiatan industri, seperti untuk kebutuhan pakan, pangan, energi, hingga pembuatan kertas (Chen, Ko, Huang & Guo, 2015;Comfort et al, 2004;Dhawan & Kaur, 2007;Khasin, Alchanati & Shoham, 1993).…”
Section: Pendahuluanunclassified
“…pakan ternak, produksi biofuel, pembuatan bir (Chen et al, 2015), dan industri pulp dan kertas sebagai katalis dalam proses pemutihan (pulp bleaching) (Khasin et al, 1993).…”
Section: Optimization Of Ph and Temperature Of Xylanolytic Isolatesunclassified
ABSTRAKBakteri laut penghasil enzim xilanase dan mananase menyimpan banyak potensi bagi bioteknologi kelautan. Informasi sebarannya juga dapat digunakan untuk memetakan keragaman bakteri laut serta analisis lingkungan perairan Indonesia sehingga pemanfaatannya dapat tepat sasaran. Penelitian ini bertujuan untuk mengisolasi bakteri laut pendegradasi xilan dan manan dari Indonesia. Penapisan kemampuan xilanolitik dan manolitik menggunakan metode Congo red dilakukan pada isolat bakteri yang diisolasi dari Laut Jawa, Selat Makassar, Laut Flores, dan Laut Sawu pada kedalaman 5 dan 20 m. Hasil penelitian menunjukkan bahwa isolat X7517, X1654, dan XM26511 diketahui menghasilkan xilanase dengan kondisi optimal reaksi enzim masing-masing adalah pH 9, T 90 °C (2,253±2,075 U/ml), pH 6, T 70 °C (0,633±0,082 U/ml), dan pH 6, T 70 °C (2,293±0,066 U/ml). Ketiganya diketahui memiliki kesamaan genetis dengan Halomonas aquamarina DSM 30161, Alteromonas macleodii NBRC 102226, dan H. meridiana NBRC 15608. Isolat bakteri manolitik L15203 dan L16571 memiliki kesamaan dengan Idiomarina zobellii KMM231, sedangkan isolat L2207 memiliki kesamaan dengan Bacillus sp. MB 71. Ketiga bakteri tersebut memiliki aktivitas mananase optimal pada kondisi alkali, masing-masing pada pH 8, T 80 °C (0,477±0,024 U/ml) untuk L 15203, pH 9, T 90 °C (0,476±0,009U/ml) untuk L16571 dan pH 9, T 80 °C (0,528±0,057 U/ml) untuk L2207. Bakteri laut xilanolitik dan manolitik yang berpotensi dalam produksi xilanase dan mananase melimpah di sebagian perairan Indonesia, terutama di perairan yang semakin dekat permukaan laut atau perairan dangkal. 8, T 70 °C (0.477±0,024 U/ml), pH 9, T 80 °C (0.476±0,009U/ml) and pH 9, T 80 °C (0.528 ±0,057 U/ml), respectively. Xylanolytic and mannolytic marine bacteria that are potential for xylanase and mannanase production were found abundantly on parts of Indonesian waters particularly at shallow water.
KATA KUNCI: bakteri laut Indonesia, xilanase, mananase
ABSTRACT
Xylanase and mannanase producing marine bacteria are potential to be utilized in marine biotechnology. Information of their distribution is important to determine the biodiversity pattern of marine bacteria and to analyze the aquatic environment in
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