Heterologous Expression Of Three Glycoside Hydrolases And Their Use Of Improveing Tea Aroma

Glycoside hydrolase is a kind of hydrolase that catalyzes the breaking of glycosidic bond of glycosides,which was widely used in food flavor improvement.Aroma is an essential quality of tea,and plenty of glycoside aroma precursors exist in tea products.Our previous study found that the crude extracellular enzyme of Aspergillus niger increased the volatile aroma components in tea product.To further determind the related glycosidases involved in the transformating of glycoside aroma precursors,A.nigerβ-glucosidase,β-xylosidase andα-L-arabinosidase were heterologously expressed in Pichia pastoris.Volatile compounds of instant Oolong tea powder treated by these three glycosidases were qualitatively and quantitatively analyzed by GC-MS.The main results were as follows:1)Aβ-glucosidase（An-Glu）with molecular weight of 120 k Da was heterologously expressed.An-Glu exhibited different substrate specificities with hydrolysis of p NPG and the measured natural substrates.The optimal temperature and pH of An-Glu were 65℃ and 4.5,respectively towards p NPG.An-Glu had good thermal stability and pH stability.The half-lives of An-Glu at 60℃,65℃ was 29.4 h and 3.9 h,respectively.Over 80.0%of the maximum enzyme activity was retained after incubatiing at pH 3.0-7.0 for 24 h.An-Glu had poor glucose tolerance.The enzyme activity of An-Glu was completely inhibited by 0.1 mM glucose.Except that 10 mM Mn²⁺inhibited about 30%of the An-Glu activity,other metal ions had little effect on the An-Glu activity.Both inhibitors and surfactants could inhibite the activity of An-Glu,and SDS significantly inhibited the enzyme activity.The K_m and V_max of recombinantβ-glucosidase were1.62 mM and 84.75μmol/（min·m L）,respectively.2)Theβ-xylosidase（An-Xyl）with molecular weight of 120-150 k Da showed the dual function of hydrolyzing p NPX and p NPG.When p NPX was used as substrate,the optimal temperature and pH were 65℃ and 4.0 respectively.An-Xyl had good thermal stability and pH stability.The half-lives of An-Xyl at 65℃,70℃ was 433.2 min and 10.5 min respectively.Over80.0%of the maximum enzyme activity was retained after incubating at a wide pH range（pH 2.0-8.0）for 24 h.An-Xyl had excellent xylose tolerance.The activity of An-Xyl was increased by low concentrantion（less than 50 mM）,and it was inhibited by high xylose concentrantion（50-500mM）.400 mM xylose inhibited the enzyme activity to 58.2%.The activity of of An-Xyl were slightly promoted by the measured metal ions at 1 mM,10 mM of Na⁺,Mg²⁺,Ba²⁺increased it by more than 1-fold,and strongly inhibited by Zn²⁺,Al³⁺（about 50%）.An-Xyl was inhibited by 50%in the presence of 1 mM CTAB.High concentration of inhibitors and surfactants inhibited the enzyme activity,and 10 mM SDS completely inhibited the activity of An-Xyl.The K_m and V_max of An-Xyl were 3.6 mM and 10000μmol/（min·m L）,respectively.3)Theα-L-arabinofuranosidase（An-Ara）with molecular weight of 85 k Da was expressed.The optimal temperature and pH of An-Ara were 65℃ and 5.0,respectively.toward p NPA.An-Ara had good thermal stability and pH stability.The half-lives of it at 65℃,70℃ were 157.53min and 10.19 min,respectively.About 70%of the maximum enzyme activity were retained after incubatiing at pH 4.0-8.0 for 24 h.An-Ara had excellent arabinose tolerance.The enzyme activity of An-Ara was slightly activated when the arabinose concentration was less than 400 mM,and it was inhibited by high arabinose concentration（400-2000 mM）.1 M arabinose inhibited the enzyme activity to 75.8%.The activity of of An-Ara were strongly inhibited by 1 mM Cu²⁺,Al³⁺and 10 mM Cu²⁺,Cd²⁺,Al³⁺,Fe³⁺,Zn²⁺（about 50%-80%）,and it was activated by the rest of the metal ions.The enzyme activity of An-Ara was completely inhibited by 1 mM CTAB,and it was slightly inhibited by urea.The enzyme activity of An-Ara was slightly activated by SDS and low concentration of Tween-20.High concentration of inhibitors and surfactants inhibited the enzyme activity.The K_m and V_max of An-Ara were 2.31 mM and 625μmol/（min·m L）,respectively.4)The effects of three glycosidases on volatile components in instant oolong tea powder were analysed by GC-MS.A total of 44 volatile components were identified,afterβ-glucosidase treatment,including 15 alcohols,7 aldehydes,6 esters,8 olefins,5 ketones and 3 others.After treated withβ-glucosidase,five new volatile compounds,cis-3-hexenol,1-hexanol,2-heptanol,3-octanol and benzaldehyde were generated from instant oolong tea powder.Meanwhile,the contents of benzyl alcohol,octanol,phenylethanol,nonanol,nerol,geraniol and methyl salicylate also increased.The enzyme could increaseds the volatile components of instant oolong tea powder.After separate treatment withβ-xylosidase andα-L-arabinofuranosidase,the volatile components in instant oolong tea powder were similar to those in the original tea powder.A total of 44 volatile components were identified,including 11 alcohols,6 aldehydes,6 esters,8 olefins,5 ketones and3 others.The volatile components of the instant oolong tea powder treated with complex enzymes were similar to the results ofβ-glucosidase treatment alone.Thus,the expressed recombinantβ-glucosidase could increase the aroma in the instant oolong tea powder.Due to its different substrate specificities and good stability,theβ-glucosidase would be used as an important tool enzyme for biotransformation of other glycoside precursors.The expressedβ-xylosidase andα-L-arabinofuranosidase had no effect on the aroma of tea,but they have good monosaccharides tolerance and stability,and has strong potential in cellulose degradation and juice clarification.This study had enriched the resources of glycoside hydrolases and provided a reference for the subsequent exploration of the application value of these glycoside hydrolases.it had confirmed that theβ-glucosidase An-Glu of Aspergillus niger was a related exogenous enzyme involved in the formation of volatile components,which provided a theoretical and research basis for the use of exogenous enzymes to improve the aroma of instant tea powder and to explore the formation mechanism of tea aroma quality.