Molecular Cloning and Characterizations of Xylanase Inhibitor Protein from Wheat (Triticum Aestivum)

Abstract

Xylanase inhibitor proteins (XIPs) were regarded to inhibit the activity of xylanases during baking and gluten-starch separation processes. To avoid the inhibition to xylanases, it is necessary to define the conditions under which the inhibition takes place. In this study, we cloned the XIP gene from 2 different variety of Triticum aestivum, that is, Zhengmai 9023 and Zhengmai 366, and investigated the properties of XIP protein expressed by Pichia pastoris. The results showed that the 2 XIP genes (xip-9023 and xip-366) were highly homologous with only 3 nucleotide differences. XIP-9023 showed the optimal inhibition pH and temperature were 7 °C and 40 °C, respectively. Inhibition of xylanase by XIP-9023 reached the maximum in 40 min. At 50% inhibition of xylanase, the molar ratio of inhibitor: xylanase was 26:1. XIP-9023 was active to various fungal xylanases tested as well as to a bacterial xylanase produced by Paenibacillus sp. isolated from cow rumen.

Characteristics of a XIP‐resistant xylanase from Neocallimastix sp. GMLF1 and its advantage in barley malt saccharification

“…As a previously confirmed XIP‐I sensitive xylanase (Liu et al ., ), ThXyn1 from Trichoderma harzianum was selected for a comparison study with a XIP‐I resistant xylanase. ThXyn1 in this study was identified to be the same as a UniProt protein P48793 by using MALDI‐TOF/TOF mass spectrometry, which generated four peptides (VINFSGSYNPNGNSYISI, LGEVTSDGSVYDIYR, CRVNQPSIIGTATFYQY and VNQPSIIGTATFYQYWSY) whose amino acid sequences were among P48793 sequence.…”

“…The GH11 xylanase produced by Trichoderma harzianum , here designated as ThXyn1, was previously confirmed to be sensitive to XIP‐I inhibition (Liu et al ., ). The commercial xylanase from T. harzianum (Sunson, China) was used for ThXyn1 preparation.…”

“…In our previous work, the gene of XIP‐I (named xip ‐9023) from T. aestivum Zhengmai 9023 was expressed in Pichia pastoris GS115 through pPIC9K vector, and the pure XIP‐I protein was obtained by simply collecting the secreted proteins of the recombinant P. pastoris strain (Liu et al ., ). Here, the XIP‐I was prepared by the same way.…”

“…The inhibition assay of XIP‐I towards xylanases was performed as described previously with some modification (Liu et al ., ). Briefly, 50 μL of properly diluted xylanase (xylanase concentration was approximately 1.8 IU mL −1 ) was mixed with 50 μL of XIP‐I (2.5–3.0 μ m ), and the mixture was incubated at 30 °C for 30 min and then the residual xylanase activity was measured as described above, with the same amount of xylanase mixed with the inactivated XIP‐I (boiled for 5 min in advance) as a control.…”

Barley α‐amylase/subtilisin inhibitor shows inhibitory activity against endogenous xylanase isozyme I of malted barley: A novel protein function

“…Debyser, Derdelinckx, and Delcour (1997a) first reported that wheat ( Triticum aestivum ) contained proteinaceous xylanase inhibitor which inhibited the xylanolytic system of malted barley. So far, three proteinaceous xylanase inhibitors have been purified and characterized from cereals, including Triticum aestivum xylanase inhibitor (TAXI) (Leys, De Bondt, Schreurs, & Courtin, 2019), thaumatin‐like xylanase inhibitor (TLXI) (Denisenko et al, 2019), and xylanase‐inhibiting protein (XIP) (Liu, Zhang, Wei, Chen, & Jia, 2017). The reported proteinaceous xylanase inhibitors hereto in cereals are active toward microbial xylanases, and no proteins have been reported to have the ability to inhibit the catalytic activity of the cereal endogenous xylanases (Denisenko et al, 2019).…”