Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids

Abstract

XynB from Aspergillus niger ATCC1015 (AnXynB) is a mesophilic glycoside hydrolase (GH) family 11 xylanase which holds great potentials in a wide variety of industrial applications. In the present study, the catalytic activity and stability of AnXynB were improved by a combination of computational and experimental approaches. Virtual mutation and molecular dynamics simulations indicated that the introduction of Glu and Asn altered the interaction network at the - 3 subsite. Interestingly, the double mutant S41N/T43E displayed 72% increase in catalytic activity when compared to the wild type (WT). In addition, it also showed a better thermostability than the WT enzyme. Kinetic determination of the T43E and S41N/T43E mutants suggested that the higher xylanase activity is probably due to the increasing binding affinity of enzyme and substrate. Consequently, the enzyme activity and thermostability of AnXynB was both increased by selective site-directed mutagenesis at the - 3 subsite of its active site architecture which provides a good example for a successfully engineered enzyme for potential industrial application. Moreover, the molecular evolution approach adopted in this study led to the design of a library of sequences that captures a meaningful functional diversity in a limited number of protein variants.

Characterization of a novel xylanase from Aspergillus flavus with the unique properties in production of xylooligosaccharides

“…Similar to the xylanase from A. niger [23], the AfXynB showed its highest activity in a neutral pH (pH 7.5), while most other reported xylanases were active in a weakly acidic pH ranged from 5.0 to 7.0 [19,22,25]. Besides, the AfXynB was found stable to retain more than 82.3% of its initial activity within pH 4.0-9.5, which is similar to the crude xylanase from A. nidulans [20].…”

“…since they are capable of producing high levels of extracellular enzymes and can be cultivated very easily [20]. Although a great number of xylanases have been studied in different Aspergillus strains [21][22][23], few reports are available on the properties of the xylanases from A. flavus. Till date, only four xylanases from A. flavus had been purified and characterized, which had respective molecular mass at 14.0, 20.2, 28.5, and 35.0 kDa [24][25][26][27].…”

A Sequence Embedding Method For Enzyme Optimal Condition Analysis

“…For these purposes, biological researchers generally perform a series of gradient tests to measure the optimal condition of each enzyme in a family. Then they adopt the protein engineering to produce an enzyme with an expected optimal condition [3]. But the above biological methods analyze only an enzyme at each wet experiment such that they cost much time, power and resources to get the expected enzyme.…”

“…We crawl the amino acid sequences of the GH11 family on the CAZy website [3] . In order to extract the required optimal pH of each sequence, we investigate the papers related to the proteins in the GH11 family on the W eb of Science website.…”

“…Since the size of a family dataset is small, we adopt the approximation method to generate more samples by the statistics on amino acids in the training dataset. To understand how this method works, we compare the probability distribution on [3] The CAZy database describes the families of structurally-related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds. http://www.cazy.org/ the training samples and all samples by the KL distance, an often adopted metric to evaluate two probability distributions [15],…”