The Functional And Molecular Properties Of Xylosidase JB13GH39

Xylans are the highest content of hemicellulose and abundant in many agricultural biomasses,algae,and industrial wastes.?-xylosidases produce xylose from xylans after degradation by endoxylanases.Xylose can be utilized to produce xylitol,lactic acid,bioethanol,and other chemicals by engineered Saccharomyces cerevisiae.It is important to maintain activity in low-pH,medium to low temperatures or high-ethanol conditions for enzymes during the process.The properties of low-temperature activity,salt and protease tolerance of?-xylosidase in the process of making bread,saline foods and other manufactured food are useful.Certain?-xylosidases can transfer xylosyl moieties to specific acceptors or remove the xylosyl group from notoginsenosides R₁ and R₂ to yield ginsenosides Rg₁ and Rh₁,respectively.The products have various potential applications.For example,the ginsenosides Rg₁ and Rh₁ possess the function of anticancer,antioxidant,and anti-inflammatory.In conclusion,the?-xylosidase with the above functional properties has great potential in the food,feed and pharmaceutical industries.Studying the molecular characteristics can promote the understanding of the mechanism corresponding to the functions,and thus can guide the molecular design of enzymes.That is of important theoretical research value.At present,the?-xylosidase having the above functional properties and its mechanism of action have not been reported yet.We obtained a new strain Sphingomonas sp.JB13 in our laboratory previously.And we have studied exo-inulinase,galactosidase and mannanase from the strain,which all have multifunctional characteristics such as low-temperature activity and ethanol tolerance.There is a novel GH39 enzyme in the genome of JB13.The sequence of the GH39 enzyme has the highest homogeneity of 75.9%with the sequences in the database.The protein was found to have?-xylosidase activity by substrate specificity analysis.Based on the novelty of strain JB13,the novelty of functions of other enzymes from strain JB13,and the novelty of the xylosidase sequence,it is speculated that the xylosidase should have novel and special functions and molecular characteristics.It is urgent to study it.Therefore,the functional or molecular properties were studied as follows:1.Investigating the functional properties of?-xylosidase JB13GH39The?-xylosidase gene jb13GH39 was ligated into pEASY-E2 vector,then transformed into E.coli for expression and purification to obtain recombinant enzyme rJB13GH39.rJB13GH39 has the following functional properties:rJB13GH39 is optimal at pH 4.5 and stable at pH 4.0-9.0;The optimum temperature is 50°C and the enzyme retained 10%-50%of its maximum activity at 0-20°C.Most salts and chemical reagents including 3.0%-20.0%?w/v?NaCl showed little or no effect on the enzymatic activity.rJB13GH39 exhibited 71.9%and 55.2%activity in 10.0%and 15.0%?v/v?ethanol,respectively.rJB13GH39 was stable in3.0%-30.0%?w/v?NaCl,3.0%-20.0%?v/v?ethanol,and 2.2-87.0 mg/mL trypsin.The enzyme transferred one xylosyl moiety to certain sugars or alcohols to form new functional products,such as xylose-glucose and xylose-alcohol.The enzyme had the ability to hydrolyze oPNX and notoginsenosides R₁ and R₂ to ginsenosides Rg₁ and Rh₁ respectively.2.Investigating the molecular features of?-xylosidase JB13GH39.Analysing and comparing sequences and structures of JB13GH39 and other GH39?-xylosidases found that JB13GH39 had high structural flexibility caused by high proportions of small amino acids ACDGNSTV and random coils.Finally,the catalytic energy-barrier is reduced,more over,internal hydrophobic interaction and electrostatic interaction of JB13GH39 are weakened.There are the moleculars mechanism of the activity of low-temperatures as well as salt-ethanol tolerence.rJB13GH39 can convert notoginsenoside R₁ and R₂ to produce ginsenoside Rg₁and Rh₁,respectively,because the enzyme can hydrolysis?-1,2-glycosidic bond.Specifically,rJB13GH39 cleavages?-1,2-glycoside bond between xylose and glucose.To study the molecular basis of its?-1,2-xylosidase activity,the results of analysis for enzyme-ligand docking with xylose-glucose linked by?-1,2-glycoside bond as a ligand revealed that seven amino acid sites bound to the ligand with hydrogen bond frequently.?-1,2-xylosidase activity is not the specific activity of JB13GH39,and some other GH39?-xylosidase reported have this activity.So the conserved site analysis of 14 GH39?-xylosidases that have been reported to have?-1,4-xylosidase or?-1,2-xylosidase activity revealed seven conserved amino acid sites involved in?-1,2-xylosidase activity,five of which were invariant and two could be changed.The two variable positions are Ps266 and Ps322,which is Tyr257and Tyr311 that have a lower hydrogen binding frequency in the docking of JB13GH39 and xylose-glucose,respectively.Furthermore,phylogenetic analysis,conserved sequence sites and enzyme-ligand docking analysis of JB13GH39 speculated that GH39?-xylosidase owning?-1,4-xylosidase activity should have?-1,2-xylosidase activity.The two variable amino acid positions Tyr257 and Tyr311 were mutated to threonine?Thr?and proline?Pro?,respectively.And two mutants still had?-1,4-xylosidase and?-1,2-xylosidase activity,but the activity was reduced.In addition,in the classification of enzymes by the International Committee of Enzymes,?-1,2-xylosidase activity has not been recorded.?-1,2-xylosidase activity should be classified as an EC number and represented by GH39?-xylosidases.In summary,?1?this study obtained a novel and multifunctional?-xylosidase,which showed salt/ethanol/trypsin tolerance,low-pH/low-temperature activity,transxylosylation and?-1,2-xylosidase activity.The enzyme can be used in food,brewing,bioenergy and pharmaceutical industries.It is especially suitable for the saccharification and fermentation using S.cerevisiae to produce ethanol,the preparation of xylose-alcohols,and the conversion of notoginsenosides.?2?The enzyme has high frequency of small amino acids and random coils,which enhance its structural flexibility and result in salt/ethanol tolerence and low temperature activity.?3?The molecular properties relate to activity of transformation for notoginsenoside or?-1,2-xylosidase.The study proposed that all GH39?-xylosidases should have?-1,2-xylosidase activity.The activity should be classified as a new hydrolase subclass and involves seven conserved amino acid sites?His88,Glu189,Tyr257,Glu306,Tyr311,Typ344,and Glu352?.The analysis of the above molecular properties provides a theoretical basis for the molecular design and modification of xylosidases,and improves the understanding of the mechanisms regarding enzyme functions.