| Xylooligosaccharides,as super prebiotic concerned and praised since the new century,have the high quality functions of enhancing immunity,reducing blood pressure and blood lipid,regulating intestinal flora and improving food flavor.At present,xylo-oligosaccharides are mainly produced by alkali extraction of corncob and further enzymatic hydrolysis.This method consumes a lot of acid and alkali liquor,and has a heavy environmental burden.The objective of this study is to explore the green and environmental enzymatic preparation of xylooligosaccharides.When wheat bran was used as raw material to prepare xylooligosaccharides,ester bonds still exist on the side chains of xylan because the expected preparation process does not involve alkali extraction,while acetyl xylan esterase plays a good assistant role in enzymatic hydrolysis.In this study,an acetyl xylanase AXE+CBM1 with CBM1 domain was cloned from Talaromyces leycettanus JCM12802.In order to explore the role of CBM1,the full-length and truncated acetylxylanase were successfully expressed in Pichia pastoris.When AXE and AXE+CBM1 were combined with NPXYN11,a xylanase from Neocallimastix patriciarum,respectively,to starch-free wheat bran,the amount of reducing sugars increased by 9% and 13% compared with that of NPXYN11 alone,suggesting that acetyl xylan esterase was beneficial to the degradation of starch-free bran by xylanase,and the presence of CBM1 made it more effective.Similarly,we introduced the CBM1 domain into xylanase NPXYN11 and successfully expressed in Pichia pastoris,enzymatic characterization showed that the presence of CBM1 had little effect on it.In the experiment of degrading starch-free bran,the higher the concentration of substrates,the stronger the effect was.Acetyl xylan esterase first reacts with xylan and eliminates acetyl groups,which can better promote the role of xylanase.At the same time,the combination of AXE+CBM1 and NPXYN11+CBM1 increased the reducing sugar by nearly 35% compared with NPXYN11 and AXE,and the proportion of xylooligosaccharides component remained unchanged,most of them(86%)were 49% xylobiose and 37% xylotriose.Wood pulp waste liquor has abundant xylan,is a waste resource of paper industry and also a good raw material for preparing xylooligosaccharides.Compared with the acid-base precipitation method,which is pressured on the environment at present,the green method of preparing xylooligosaccharides by bio-enzymatic degradation is more worthy of study and praise.Previous studies have shown that high temperature is more conducive to product transformation.Based on literature reports,we chose a thermophilic GH11 family xylanase EVXYN11 to express in Escherichia coli and Pichia pastoris.The characterization showed that the recombinant enzyme expressed by yeast was significantly weaker than that expressed in Escherichia coli in terms of thermal stability.It was speculated that glycosylation caused a decrease in thermal stability during yeast expression.Three potential N-glycosylation sites were mutated.The results showed that the glycosylation site of N135 on thumb Loop was directly related to thermal stability.The super heat-resistant mutant N135 T was obtained by saturation mutation study.When beechwood xylan was used substrate,the catalytic efficiency increased by 1.23 times and the enzyme activity remained stable for a long time at high temperature(100% and 84% of the remaining enzyme activity at 70,80 °C for 8 h).In particular,the amount of reducing sugar produced by N135 T mutant degrading wood pulp effluent was 2.7 times that of wild type and 3.5 times that of commercial enzyme NPXYN11.The analysis of products showed that the total amount of xylobiose and xylotriose could reach 85%.In this study,the synergistic degradation of wheat bran by acetyl xylan esterase and xylanase,and the conversion of super heat-resistant xylanase into wood pulp waste liquor were systematically studied,which provided a good idea for green production of xylooligosaccharides. |
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