Construction Of Thermostable β-1,3-1,4-glucanase And Its Enzymatic Properties

β-1,3-1,4-glucanases （E.C.3.2.1.73, β-glucanases） are glycosyl hydrolases, which exhibit strict substratespecificity for cleavage of β-1,4glycosidic bonds in3-O-substituted glucopyranose units. β-glucanases are animportant industrial enzyme widely used in beer and feed industries. In the brewing industry, barley β-glucan cancaused severe problems such as reduced yield of extract and lowered rates of wort separation or beer filtration,and causing the precipitation during beer storage. In anminal feed, β-glucan will increase the viscosity of feed, andeffect the contaction between endogenous digestive enzyme and nutrients, and reduce the nutritional value of feed.β-glucanases can effectively avoid the negative impact of grain β-glucan in brewing and feed industries andimprove the quality of beer and the biological efficiency of feed.The major drawback of the current β-glucanases is their poor thermal stability and low activity. β-glucanasefrom Bacillus amyloliquefaciens displays high activity under acidic conditions, but has poor thermal stability.Clostridium thermocellum is a thermophilic anaerobic bacterium, and its β-glucanase displayed higher thermalstability than Bacillus β-glucanase, while β-glucanase from Clostridium thermocellum has low activity. In thisstudy, constructing a β-glucanase with high thermal stability and catalytic activity is the major purpose. Theβ-glucanase genes from Bacillus amyloliquefaciens and Clostridium thermocellum were chosen as reserech object,fusion enzymes were constructed between them using gene splicing by overlap extension technology. The mainfindings are as follows:1. β-glucanase from Bacillus amyloliquefaciens was constructed. The activity of β-glucanase in thefermentation broth was80U mL^-1, specific activity of recombinant enzyme was1106U mg^-1after purification,and the recombinant enzyme exhibited maximal activity at50℃and pH6.0. The Michaelis constants （K_m） andcatalytic efficiency (K_cat/K_m) of recombinant enzyme were1.5mg mL^-1and369mL mg^-1s^-1, respectively. Thethermal stability of recombinant enzyme was not high, which only retained16%of activity following incubationat80℃for30min.2. β-glucanase deleted dockerin domain from Clostridium thermocellum was constructed. The activity ofβ-glucanase in the fermentation broth was7.8U mL^-1, Specific activity of the recombinant enzyme was275Umg^-1after purification, the optimal temperature and pH of the recombinant enzyme were70℃and8.0,respectively. The enzyme displayed high stability between pH7.0and9.0. Compared with β-glucanase fromBacillus amyloliquefaciens, the recombinant enzyme displays low catalytic activity, but has high thermal stability.The Michaelis constants （K_m） and catalytic efficiency (K_cat/K_m) of recombinant enzyme were2.7mg mL^-1and51mL mg^-1s^-1, respectively. When incubated at80℃for30min, the recombinant enzyme can retain60%residualactivity.3. The fusion β-glucanases were constructed by end-to-end fusion between N-terminal of the β-glucanasefrom Bacillus amyloliquefaciens and the N-terminal13or27amino acid fragments of β-glucanase fromClostridium thermocellum. The fusion β-glucanases were named as R13and R27, respectively. Specific activity ofR13and R27were1074U mg^-1and1013U mg^-1after purification, respectively. The optimal temperature of R13and R27was all60℃, and the optimal pH was all6.0. R13and R27can retain34%and52%of their activityfollowing incubation at80℃for30min, which was18%and36%higher than that of β-glucanase from Bacillusamyloliquefaciens, respectively, but they all displayed lower thermal stability than that of β-glucanase fromClostridium thermocellum. The result showed that β-glucanase from Bacillus amyloliquefaciens fused with theN-terminal13or27amino acid fragments of β-glucanase from Clostridium thermocellum, can significantlyimp-rove its thermal stability. The Michaelis constants （K_m） and catalytic efficiency (K_cat/K_m) of R13were1.7mgmL1and324mL mg^-1s^-1, respectively, while K_mand K_cat/K_mof R27were1.9mg mL^-1and288mL mg^-1s^-1,respectively. Compared with the catalytic activity of β-glucanase from Bacillus amyloliquefaciens, R13and R27displayed low catalytic activity, because of its low the affinity with substrate, and β-glucanase from Bacillusamyloliquefaciens constructed with27amino acid fragment in which its enzymatic activity inhibition greater than13amino acid fragment.4. β-glucanase with double catalytic domain was constructed by end-to-end fusion of catalytic domain ofβ-glucanase from Bacillus amyloliquefaciens and Clostridium thermocellum. β-glucanase with double catalyticdomains was named as RQ. Specific activity of RQ was2434U mg^-1afeter purification, the optimal temperatureand pH of RQ was70℃and6.0, respectively. At80℃incubation for30min, RQ can retain67%residualactivity, which was33%and15%higher than that of R13and R27, respecitively. The thermal stability of RQ wassimilar with that of β-glucanase from Clostridium thermocellum. K_mand K_cat/K_mof RQ were1.2mg mL^-1and1014mL mg^-1s^-1, respectively. The catalytic efficiency of RQ was3.13and3.52-fold higher than that of R13andR27, respectively, which was also2.41-fold higher than that of superposition between Bacillus amyloliquefaciensand Clostridium thermocellum. It showed that RQ was a new enzyme, whose enzymatic properties were notsimple superposition between β-glucanase from Bacillus amyloliquefaciens and Clostridium thermocellum, butgiven the new enzymatic properties. The thermal stability of RQ was similar with that of β-glucanase from Clostridium thermocellum, and whose the catalytic activity was also much higher than that of superposition ofβ-glucanase from Bacillus amyloliquefaciens and Clostridium thermocellum. The linker of β-glucanase fromClostridium thermocellum can effectively improve the thermal stability and catalytic activity of RQ.5. In order to study the heat-resistant mechanism of RQ, A site-directed mutant enzyme of RQ as RQM wasconstructed, in which two key glutamate residues （E134and E138） in active site of β-glucanase from Clostridiumthermocellum were reconstructed by substitution of alanine （E134A/E138A）. The thermostable study showed thatRQM can retain57%of activity following incubation at80℃for30min, which was41%,23%and5%higherthan that of β-glucanase from Bacillus amyloliquefaciens, R13and R27, respectively. And the thermal stability ofRQM was3%and10%lower than that of β-glucanase from Clostridium thermocellum and RQ, respectively.These results suggest that the thermal stability of RQ was not only with N-terminal of β-glucanase fromClostridium thermocellum, but also involves all of the enzyme molecules, including its catalytic sites. The newβ-glucanase with fine features was constructed between β-glucanase from Bacillus amyloliquefaciens andClostridium thermocellum, in which the catalytic domain of β-glucanase from Clostridium thermocelluminteracted with that of β-glucanase from Bacillus amyloliquefaciens, and resulting in synergy effects.6. The optimization of fermentation medium and conditions for RQ were investigated. The results indicatedthat the highest production of β-glucanase could be obtained in the medium containing (g L^-1): barley powder43.48, soybean powder34.40, NaCl2.40, KH₂PO₄2.40and K₂HPO₄12.50. And the optimal fermentationconditions are as follows: inoculation volume1%（V/V）, loading volume45mL/250mL, initial pH6.0-7.0,induced after cultivating for3h at200rpm. Compared with the initial fermentation medium, the product ofβ-glucanase cultivated by optimized fermentation medium increased by11%, which was110U mL^-1. Althoughthe production of β-glucanase didn’t get a substantial improvement, barley powder and soybean powder werechosen as carbon and nitrogen sources in optimization fermentation medium, which are cheap meterials. All ofthis can significantly reduced the costs of β-glucanase, and has good practical value.