Gene cloning, expression, immobilization and characterization of endo-xylanase from Geobacillus sp. TF16 and investigation of its industrial applications

Q2 Chemical Engineering

Journal of Molecular Catalysis B-enzymatic Pub Date : 2016-11-01 DOI:10.1016/j.molcatb.2017.01.016

Ummuhan Cakmak, Nagihan Saglam Ertunga

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引用次数: 19

Abstract

The xylanase gene (xynTF16) from a thermophilic bacterium Geobacillus sp. TF16 was cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant enzyme (GsXynTF16) was purified 52-fold by nickel affinity chromatography, and determined as a single band 39.8 kDa on SDS-PAGE with a specific activity of 246 U/mg protein. The recombinant enzyme was immobilized on chitosan with a yield of 85.6%. The enzyme showed the highest activity towards xylan. The immobilized enzyme displayed an increase in optimum temperature from 55 to 65 °C in comparison with free enzyme. While the free enzyme was optimally active at pH 8.5, immobilized enzyme showed higher activity in the pH range 6.0–8.5. Thermal and pH stability of immobilized enzyme was determined to be higher than that of the free enzyme. Immobilized xylanase could be reused for 6 consecutive cycles retaining 80% of its initial activity. It was also found to be effective in releasing the reducing sugar from juice and poultry feed and oven spring in bakery. These results suggest that this study provides an alternative xylanase enzyme with enhanced properties.

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Geobacillus sp. TF16内切木聚糖酶基因的克隆、表达、固定化、鉴定及工业应用研究

将嗜热细菌Geobacillus sp. TF16的木聚糖酶基因(xynTF16)克隆到pET-28a(+)载体中，并在大肠杆菌BL21(DE3)pLysS中表达。重组酶(GsXynTF16)经镍亲和层析纯化52倍，在SDS-PAGE上确定为39.8 kDa单带，比活性为246 U/mg蛋白。将重组酶固定在壳聚糖上，产率为85.6%。该酶对木聚糖的活性最高。与游离酶相比，固定化酶的最适温度从55℃提高到65℃。游离酶在pH为8.5时活性最佳，固定化酶在pH为6.0 ~ 8.5时活性较高。结果表明，固定化酶的热稳定性和pH稳定性均高于游离酶。固定化木聚糖酶可连续重复使用6个周期，保留初始活性的80%。对果汁、家禽饲料、烘箱弹簧中的还原糖也有较好的脱除效果。这些结果表明，本研究提供了一种性能增强的替代木聚糖酶。

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来源期刊

Journal of Molecular Catalysis B-enzymatic 生物-生化与分子生物学

CiteScore

2.58

自引率

0.00%

发文量

审稿时长

3.4 months

期刊介绍： Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation. Papers should report novel and significant advances in one or more of the following topics; Applied and fundamental studies of enzymes used for biocatalysis; Industrial applications of enzymatic processes, e.g. in fine chemical synthesis; Chemo-, regio- and enantioselective transformations; Screening for biocatalysts; Integration of biocatalytic and chemical steps in organic syntheses; Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies; Enzyme immobilization and stabilization, particularly in non-conventional media; Bioprocess engineering aspects, e.g. membrane bioreactors; Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification; Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity; Biomimetic studies related to enzymatic transformations.