N端二硫键对11家族木聚糖酶热稳定性的影响

以来源于米曲霉Aspergillus oryzae的11家族常温木聚糖酶AoXyn11A为母本,将其N端替换成同一家族耐热木聚糖酶EvXyn11TS的对应片段,构建出耐热杂合木聚糖酶AEx11A。将AoXyn11A和AEx11A基因分别在毕赤酵母GS115中进行表达并分析比较温度对表达产物酶活性的影响。结果表明,AEx11A的最适温度Topt为75℃,在70℃的半衰期t1/270为197 min,较AoXyn11A(Topt=50℃,t1/270=1.0 min)显著提高。通过对AEx11A结构的同源建模及其与AoXyn11A结构的比对,发现在AEx11A的N端引入了一个二硫键(Cys5–Cys32)。利用定点突变法将其5位的半胱氨酸突变为苏氨酸(C5T),去除该二硫键,以探讨其对AEx11A热稳定性的影响。分析表明,突变酶(AEx11AC5T)的Topt由突变前的75℃降为60℃,其t1/270和t1/280也分别由197 min和25 min缩短为3.0 min和1.0 min。     

Y13F定点突变改良米曲霉中温木聚糖酶的耐热性

为改良米曲霉（Aspergillus oryzae）糖苷水解酶11家族木聚糖酶AoXyn11A的耐热性,将其Tyr¹³（Y13）置换为Phe（F）。基于AoXyn11A与同一家族7种耐热木聚糖酶一级结构的多序列同源比对及其三维结构的同源建模和分子动力学模拟,设计了一种突变酶AoXyn11A^Y13F;以重组质粒pPIC9K-Aoxyn11A为模板,采用PCR技术将AoXyn11A基因（Aoxyn11A）中编码Y13的密码子TAC突变为F的TTC,构建了一种突变酶基因（Aoxyn11A^Y13F）;分别将Aoxyn11A和Aoxyn11A^Y13F在毕赤酵母（Pichia pastoris）GS115中实施了表达,并对重组表达产物AoXyn11A和AoXyn11A^Y13F的耐热性进行了分析。结果表明：突变酶的最适温度T_opt由突变前的50℃提高到55℃;AoXyn11A^Y13F在50℃的半衰期t_1/2⁵⁰为95 min,较AoXyn11A（t_1/2⁵⁰=6 min）延长了约15倍。由此经Y13F定点突变显著改良了野生型木聚糖酶的耐热性。     

通过N端替换提高木聚糖酶的热稳定性

以来源于Thermomonospora fusca的耐高温木聚糖酶TfxA和来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB为亲本,构建出耐热高比活融合木聚糖酶TB,将TB在大肠杆菌BL21和毕赤酵母GS115中进行表达并对表达产物的酶学性质进行分析比较。分析表明,融合蛋白TB最适pH值为6.0,最适温度为70℃,较XYNB有大幅度的提高;在热稳定性方面,TB明显优于XYNB,将两种稀释好的酶液分别在80℃和90℃下热处理3min,TB的热稳定性较XYNB提高了6倍左右;TB的pH稳定性为5～9(相对剩余活性在50%以上的pH范围),较XYNB有所下降,但两者的比活性基本不变,保持了亲本XYNB的高比活性。通过同源建模和序列比较,分析了可能影响融合蛋白TB酶学性质的因素,为进一步研究木聚糖酶的结构与功能提供了新的思路。     

N13D、S40E点突变提高木聚糖酶XYNB的热稳定性

对来源于Streptomyces olivaceoviridis的高比活木聚糖酶XYNB进行同源建模和序列比较,设计了N13D、S40E的定点突变,以期改善中温酶XYNB的热稳定性。突变酶N13D、S40E分别在毕赤酵母中表达,经纯化后与野生型酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,突变酶N13D和S40E在70℃处理5min,热稳定性比XYNB分别提高了24.76%和14.46%;突变酶N13D的比活性比XYNB提高了22%。在其他性质方面突变酶N13D、S40E与野生型酶XYNB基本相似。通过对木聚糖酶XYNB的定点突变,提高了该酶的热稳定性,并为结构与功能的进一步研究提供了材料。     

耐碱和耐热木聚糖酶研究进展

木聚糖酶是一种重要的工业用酶,近年来由于它在制浆和造纸工业中的特殊用途而受到广泛关注。在制浆和造纸工业中需要耐碱和耐热的木聚糖酶。要得到这样的木聚糖酶有两种办法,一是从极端环境中筛选这样的高产木聚糖酶的微生物,其二是用先进的生物技术对现有的木聚糖酶进行遗传改造,以达到耐热和耐碱的目的。     

定点突变提高木聚糖酶Umxyn10A的热稳定性

木聚糖酶是微生物半纤维素降解体系中的一种关键酶,被广泛地应用在工业中的多个领域。本研究为了提高木聚糖酶Umxyn10A (ABL73-883.1)的热稳定性,将Umxyn10A与GH 10家族4种耐热木聚糖酶进行多序列同源比对以及三维结构的同源建模分析,选定了Umxyn10A第31位氨基酸位点进行定点突变,将氨基酸Ala (A)突变为Phe (F)。分别将Umxyn10A和Umxyn10AA31F在大肠杆菌中进行重组表达,分析2种重组酶的酶学特性,结果发现,Umxyn10AA31F的最适反应温度为85℃,较野生重组酶提高了5℃;在65℃下的半衰期为105 min,较野生重组酶(15 min)提高了6倍;在70℃下突变重组酶的半衰期为15 min,较野生重组酶(5 min)提高了2倍;与此同时突变重组酶的pH耐受区间较原酶也有一定的增大。结果表明,将第31位氨基酸位点Ala突变为Phe能够显著的提高Umxyn10A的热稳定性。     

木聚糖酶XYNB的N46D突变、表达及酶学性质变化

对来源于Streptomyces olivaceoviridis的高比活木聚糖酶XYNB进行同源建模和同源序列比较,发现第11族木聚糖酶的催化结构域在β折叠股A3和B3之间存的一个保守的氨基酸位点,该位点与木聚糖酶的pH特性有关.据此设计了XYNB的N46D定点突变.将突变酶XYNBN46D在毕赤酵母中表达,表达的XYNBN46D经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明, XYNBN46D的最适pH值由5.2下降到4.2,pH稳定性也向酸性pH偏移,同时,热稳定性和最适温度也有一定的提高, 但酶的比活性显著下降.结果证实,木聚糖酶XYNB的第46位Asn与其最适pH值相关.对导致酶学性质改变的可能因素进行了分析,结果为进一步的结构与功能研究提供了资料.     

G蛋白偶联受体激酶—2的N端结构域缺失对其催化功能的影响

为揭示Ｇ蛋白偶联受体激酶（Ｇ ｐｒｏｔｅｉｎ－ｃｏｕｐｌｅｄ ｒｅｃｅｐｔｏｒ ｋｉｎａｓｅｓ，ＧＲＫｓ）的Ｎ端结构域在该酶家族磷酸化受体以及催化反应中的作用，对ＧＲＫ２的Ｎ端结构域进行了缺失突变研究。将Ｎ端结构域的ＧＲＫ２（ΔＮ－ＧＲＫ２）与谷城肽转硫酶融合表达。经过亲和纯化后，再用凝血酶酶切得到单独的ΔＮ－ＧＲＫ２蛋白。对磷酸化小肽的活力检测表明，Ｎ端结构域的缺失基本上不影响ＧＲＫ２的激酶催化     

木聚糖酶XYNB分子中折叠股B1和B2间的疏水作用对酶热稳定性的影响

对来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB进行同源建模,并结合嗜热木聚糖酶氮末端芳香族氨基酸疏水作用的结构分析,设计了XYNB的T11Y定点突变,观察XYNB分子中折叠股B1和B2的疏水作用对酶的热稳定性的影响。将突变酶XYNB′在毕赤酵母中表达,表达的XYNB′经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,XYNB′的耐热性比XYNB有明显的提高,但最适温度与原酶一样为60℃。另外,XYNB′的最适pH、Km值及比活性均有一定的改变。实验证实了木聚糖酶XYNB的氮端芳香族氨基酸之间的疏水相互作用与其热稳定性相关,为进一步的结构与功能研究提供了优良的基因材料。     

定点突变提高里氏木霉木聚糖酶 (XYN II) 的稳定性

通过定点突变的方法,在来源于里氏木霉Trichderma reesei的木聚糖酶XYN II的N-末端两个β折叠片层间添加二硫键,以提高木聚糖酶的稳定性。原酶XYN-OU和突变酶XYN-HA12 (T2C、T28C和S156F) 分别在毕赤酵母中分泌表达,突变酶与原酶纯化后进行酶学性质比较。结果表明：突变酶最适反应温度由50℃提高到60℃;在70℃的半衰期由1 min提高到14 min;最适反应pH为5.0,与原酶保持一致,但是在50℃、30 min条件下的pH稳定范围由4.0~9.0扩展到3.0~10.0。对木聚糖酶分子改良的结果反映出在β片层间添加二硫键可以有效改善酶在较高温度下三维结构的刚性,提高热稳定性。     

A unique disulfide bridge of the thermophilic xylanase syxyn11 plays a key role in its thermostability

Based on the hyperthermostable family 11 xylanase (EvXyn11^TS) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5–Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11^C5T-encoding gene, Syxyn11 ^C5T, was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11^C5T, were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11^C5T decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.     

Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability

This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.     

Engineering increased thermostability in the thermostable GH-11 xylanase from Thermobacillus xylanilyticus

Enzymatic hydrolysis constitutes an attractive strategy for biorefining of abundant, low-cost agricultural by-products such as wheat bran and straw. However, to adopt such an approach, efficient enzymes are required, in particular xylanases. To promote heat-induced disorganization of the complex cell wall network in wheat bran and thus increase enzymatic hydrolysis, we have attempted to improve the thermoresistance of a GH-11 xylanase that is already moderately thermostable. Using a previously described engineering strategy that involves the introduction of disulphide bridges, a mutant (Tx-xyl-SS3) displaying enhanced thermostability and thermoactivity was obtained. The half life at 70 degrees C (180 min) of Tx-xyl-SS3 is 10-fold greater than that of the wild type enzyme and its specific activity is almost doubled (3500 IU mg(-1)). Despite these improvements, Tx-xyl-SS3 was unsuitable for use at significantly higher reaction temperatures (i.e. 85-95 degrees C) and thus the initial objective of this study remained unaccomplished. However, unexpectedly even at the normal hydrolytic temperature (60 degrees C), Tx-xyl-SS3 was able to solubilize 50% of the wheat bran arabinoxylans, 10 points more than the wild type enzyme in parallel reactions. The data presented here show that this improvement is not directly linked to the increase in thermostability and/or thermoactivity, but rather to other unidentified changes to physico-chemical properties that may allow Tx-xyl-SS3 to better penetrate the cell wall network in wheat bran.     

Correlation between codon usage and thermostability

Variations of arginine codon usage between organisms may have important implications to thermostability. The preferential usage of AGR codons for arginine in thermophiles and hyperthermophiles implies positive error minimization, contributing to avoid mutations that could harm protein thermostability. This bias is not a mere consequence of increased G + C content, as it has been previously suggested, and may represent a new mechanism of adaptation to protein thermostability.     

Improvement of the thermostability and catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement

To improve the thermostability and catalytic activity of Aspergillus niger xylanase A (AnxA), its N-terminus was substituted with the corresponding region of Thermomonospora fusca xylanase A (TfxA). The constructed hybrid xylanase, named ATx, was overexpressed in Pichia pastoris and secreted into the medium. After 96-h 0.25% methanol induction, the activity of the ATx in the culture supernatant reached its peak, 633 U/mg, which was 3.6 and 5.4 times as high as those of recombinant AnxA (reAnxA) and recombinant TfxA (reTfxA), respectively. Studies on enzymatic properties showed that the temperature and pH optimum of the ATx were 60 degrees C and 5.0, respectively. The ATx was more thermostable, when it was treated at 70 degrees C, pH 5.0, for 2 min, the residual activity was 72% which was higher than that of reAnxA and similar to that of reTfxA. The ATx was very stable over a broader pH range (3.0-10.0) and much less affected by acid/base conditions. After incubation at pH 3.0-10.0, 25 degrees C for 1 h, all the residual activities of the ATx were over 80%. These results revealed that the thermostability and catalytic activity of the AnxA were enhanced. The N-terminus of TfxA contributed to the observed thermostability of itself and the ATx, and to the high activity of the ATx. Replacement of N-terminus between mesophilic eukaryotic and thermostable prokaryotic enzymes may be a useful method for constructing the new and improved versions of biologically active enzymes.     

Introduction of a disulfide bridge enhances the thermostability of a <Emphasis Type="Italic">Streptomyces olivaceoviridis</Emphasis> xylanase mutant

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109–C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109–C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70°C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.     

Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods

Background

Xylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from Aspergillus usamii E001, by N-terminus replacement.

Results

A hybrid xylanase with high thermostability, named AEXynM, was predicted by computational methods, and constructed by substituting the N-terminal 33 amino acids of AuXyn11A with the corresponding 38 ones of EvXyn11^TS, a hyperthermostable family 11 xylanase. Two AuXyn11A- and AEXynM-encoding genes, Auxyn11A and AExynM, were then highly expressed in Pichia pastoris GS115, respectively. The specific activities of two recombinant xylanases (reAuXyn11A and reAEXynM) were 10,437 and 9,529 U mg^-1. The temperature optimum and stability of reAEXynM reached 70 and 75°C, respectively, much higher than those (50 and 45°C) of reAuXyn11A. The melting temperature (T _m) of reAEXynM, measured using the Protein Thermal Shift (PTS) method, increased by 34.0°C as compared with that of reAuXyn11A. Analyzed by HPLC, xylobiose and xylotriose as the major hydrolytic products were excised from corncob xylan by reAEXynM. Additionally, three single mutant genes from AExynM (AExynM ^C5T, AExynM ^P9S, and AExynM ^H14N) were constructed by site-directed mutagenesis as designed theoretically, and expressed in P. pastoris GS115, respectively. The thermostabilities of three recombinant mutants clearly decreased as compared with that of reAEXynM, which demonstrated that the three amino acids (Cys⁵, Pro⁹, and His¹⁴) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.

Conclusions

This work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys⁵, Pro⁹, and His¹⁴ contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.     

Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix Patriciarum

In this study, we employed directed evolution and site‐directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far‐ultraviolet circular dichroism analysis showed that the melting temperatures (T_m) of the G201C and C60A–G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site‐directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A–G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well‐packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side‐chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself. Biotechnol. Bioeng. 2010;105: 861–870. © 2009 Wiley Periodicals, Inc.     

Improvement of alkali stability and thermostability of Paenibacillus campinasensis Family-11 xylanase by directed evolution and site-directed mutagenesis

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0–pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C-T182C/D16Y) with 10 °C increased in the optimum temperature (60–70 °C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k _cat /K _m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 °C) and high alkali (pH 9.0).