耐热β-1，3-1，4-葡聚糖酶的构建及其酶学性质研究
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摘要
β-1,3-1,4-葡聚糖酶(β-1,3-1,4-glucanase, E.C.3.2.1.73，简称β-葡聚糖酶)属于糖基水解酶类，降解β-葡聚糖中与β-1,3糖苷键相邻的β-1,4糖苷键。β-葡聚糖酶是一种重要的工业用酶，广泛应用于啤酒和饲料工业中。在啤酒生产过程中，大麦中的β-葡聚糖会降低麦芽汁和啤酒过滤速度，在啤酒贮存过程中容易引起沉淀等问题；在畜禽饲料中如含有较多的β-葡聚糖会增加食糜粘度，影响动物内源性消化酶与营养物质的接触，降低饲料的消化率。β-葡聚糖酶可有效避免β-葡聚糖在酿造和饲料工业中的上述不良影响，提高啤酒的质量和饲料的消化率。
热稳定性差和催化活性低是当前β-葡聚糖酶在工业应用中普遍存在的问题。解淀粉芽孢杆菌(Bacillusamyloliquefaciens)β-葡聚糖酶在酸性条件具有较高的酶活性，但其热稳定性较差；热纤梭菌(Clostridiumthermocellum)是一种嗜热的厌氧菌，其所产生的β-葡聚糖酶的热稳定性高于芽孢杆菌所产生的β-葡聚糖酶，但酶活力较低。本研究以热纤梭菌β-葡聚糖酶和解淀粉芽孢杆菌β-葡聚糖酶基因为研究材料，采用基因重叠延伸技术(SOE)，将二者β-葡聚糖酶基因进行杂合和融合，构建耐热、高活性的β-葡聚糖酶。研究的主要结果如下：
1.构建了解淀粉芽孢杆菌β-葡聚糖酶。经摇瓶发酵培养，该酶的酶活性为80U mL~(-1)，纯化后酶的比活力为1106U mg~(-1)，最适反应温度和pH分别为50℃和6.0，在pH5.0-7.0范围内，具有较高的酶活性；以大麦葡聚糖为底物时，该酶的米氏常数(K_m)和催化效率常数(K_(cat)/K_m)分别为1.5mg mL~(-1)和369mL mg~(-1)s~(-1)；在80℃温度下处理30min，解淀粉芽孢杆菌β-葡聚糖酶仅有16%的残余酶活，具有较低的耐热性。
2.构建了热纤梭菌缺失锚定结构域β-葡聚糖酶。经摇瓶发酵培养，该酶的酶活性为7.8U mL~(-1)，纯化后的比活力为275U mg~(-1)，最适反应温度和pH分别为70℃和8.0，在pH7.0-9.0范围内，具有较高的酶活性；该酶的K_m和K_(cat)/K_m分别为2.7mg mL~(-1)和51mL mg~(-1)s~(-1)，在80℃温度下处理30min，有60%的残余酶活；与解淀粉芽孢杆菌β-葡聚糖酶相比，热纤梭菌缺失锚定结构域β-葡聚糖酶的催化活性较低，但具有较高的热稳定性。
3.融合β-葡聚糖酶的构建。将热纤梭菌β-葡聚糖酶N-端13个和27个氨基酸残基分别与解淀粉芽孢杆菌β-葡聚糖酶N端融合，构建融合β-葡聚糖酶，分别命名为R13和R27。纯化后R13和R27的比活力分别为1074U mg~(-1)和1013U mg~(-1)，它们的最适反应温度和pH分别为60℃和6.0；在80℃温度下处理30min，R13和R27分别保留34%和52%的残余酶活，分别高出解淀粉芽孢杆菌β-葡聚糖酶残余酶活的18%和36%，但它们的耐热性均低于热纤梭菌β-葡聚糖酶。表明融合热纤梭菌β-葡聚糖酶N-端13个和27个氨基酸残基，能够提高解淀粉芽孢杆菌β-葡聚糖酶的耐热性。酶催化动力学研究表明，R13的K_m和K_(cat)/K_m分别为1.7mg mL~(-1)和324mL mg~(-1)s~(-1)，R27的K_m和K_(cat)/K_m分别为1.9mg mL~(-1)和288mL mg~(-1)s~(-1)。与解淀粉芽孢杆菌β-葡聚糖酶的催化活性相比较，R13和R27均有不同程度的降低，说明解淀粉芽孢杆菌N-端融合热纤梭菌β-葡聚糖酶N-端13或27个氨基酸残基后，降低了解淀粉芽孢杆菌β-葡聚糖酶与底物的亲和力，进而影响了酶的催化活性，其中融合热纤梭菌β-葡聚糖酶N-端27个氨基酸残基对解淀粉芽孢杆菌β-葡聚糖酶催化活性的抑制作用大于融合13个氨基酸残基的抑制作用。
4.双催化结构域β-葡聚糖酶的构建。将解淀粉芽孢杆菌β-葡聚糖酶的催化结构域和热纤梭菌β-葡聚糖酶的催化结构域连接，构建双催化结构域β-葡聚糖酶。构建的双催化结构域β-葡聚糖酶命名为RQ，纯化后RQ的比活力为2434U mg~(-1)，RQ的最适反应温度和pH分别为70℃和6.0；耐热性研究表明，在80℃温度下处理30min，RQ有67%的残余酶活，分别高出R13和R27残余酶活的33%和15%，RQ的耐热性与热纤梭菌β-葡聚糖的耐热性相接近；RQ的K_m和K_(cat)/K_m分别为1.2mg mL~(-1)和1014mL mg~(-1)s~(-1)，其催化效率分别是R13和R27催化效率的3.13倍和3.52倍，也是解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶催化效率之和的2.41倍。以上结果表明：RQ的酶学性质并不是简单的解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶的酶学性质的叠加，而是赋予了新的酶学特性的β-葡聚糖酶，它除具有热纤梭菌β-葡聚糖酶的热稳定性外，其催化效率也高于解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶的催化效率之和。热纤梭菌β-葡聚糖酶的连接子可有效改善双催化结构域β-葡聚糖酶的耐热性和催化活性。
5.双催化结构域β-葡聚糖酶的耐热机理初步研究。将RQ中热纤梭菌β-葡聚糖酶的两个催化活性位点的谷氨酸(E134和E138)定点突变为丙氨酸(E134A和E138A)，构建含点突变的双催化结构域β-葡聚糖酶，并命名为RQM。耐热性研究表明，在80℃温度下处理30min，RQM有57%的残余酶活，分别比解淀粉芽孢杆菌β-葡聚糖酶、R13和R27的耐热性提高了41%、23%和5%；与热纤梭菌β-葡聚糖酶和RQ的耐热性相比，RQM的耐热性分别降低了3%和10%。说明热纤梭菌β-葡聚糖酶不仅其N-端对RQ的耐热性有影响，而且其催化结构域的整个结构在稳定酶分子中发挥作用，包括其催化活性位点，热纤梭菌β-葡聚糖酶催化结构域和解淀粉芽孢杆菌β-葡聚糖酶催化结构域之间相互影响，产生协同效应，形成一种特性优良的、新的β-葡聚糖酶。
6.双催化结构域β-葡聚糖酶发酵培养基和培养条件如下：大麦粉43.48g L~(-1)、豆粉34.40g L~(-1)、NaCl2.40g L~(-1)、KH_2PO_42.40g L~(-1)和K_2HPO_412.50g L~(-1)；接种量1%(V/V)、装样量45mL/250mL、发酵培养基初始pH6.0-7.0、最佳诱导时期为在200rpm转速下培养3h后诱导。优化后的发酵培养基产β-葡聚糖酶的量为110U mL~(-1)，比初始发酵培养基提高了11%。虽然发酵产β-葡聚糖酶的量没有得到大幅度提高，但是优化后的发酵培养基中的碳源和氮源是廉价的大麦粉和豆粉，大大降低了β-葡聚糖酶的生产成本，具有很好的实际应用价值。
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~(-1)after 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~(-1)and369mL mg~(-1)s~(-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~(-1)after 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~(-1)and51mL mg~(-1)s~(-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~(-1)and1013U mg~(-1)after 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~(-1)s~(-1), respectively, while K_mand K_(cat)/K_mof R27were1.9mg mL~(-1)and288mL mg~(-1)s~(-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~(-1)afeter 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~(-1)and1014mL mg~(-1)s~(-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, KH_2PO_42.40and K_2HPO_412.50. 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.
热稳定性差和催化活性低是当前β-葡聚糖酶在工业应用中普遍存在的问题。解淀粉芽孢杆菌(Bacillusamyloliquefaciens)β-葡聚糖酶在酸性条件具有较高的酶活性，但其热稳定性较差；热纤梭菌(Clostridiumthermocellum)是一种嗜热的厌氧菌，其所产生的β-葡聚糖酶的热稳定性高于芽孢杆菌所产生的β-葡聚糖酶，但酶活力较低。本研究以热纤梭菌β-葡聚糖酶和解淀粉芽孢杆菌β-葡聚糖酶基因为研究材料，采用基因重叠延伸技术(SOE)，将二者β-葡聚糖酶基因进行杂合和融合，构建耐热、高活性的β-葡聚糖酶。研究的主要结果如下：
1.构建了解淀粉芽孢杆菌β-葡聚糖酶。经摇瓶发酵培养，该酶的酶活性为80U mL~(-1)，纯化后酶的比活力为1106U mg~(-1)，最适反应温度和pH分别为50℃和6.0，在pH5.0-7.0范围内，具有较高的酶活性；以大麦葡聚糖为底物时，该酶的米氏常数(K_m)和催化效率常数(K_(cat)/K_m)分别为1.5mg mL~(-1)和369mL mg~(-1)s~(-1)；在80℃温度下处理30min，解淀粉芽孢杆菌β-葡聚糖酶仅有16%的残余酶活，具有较低的耐热性。
2.构建了热纤梭菌缺失锚定结构域β-葡聚糖酶。经摇瓶发酵培养，该酶的酶活性为7.8U mL~(-1)，纯化后的比活力为275U mg~(-1)，最适反应温度和pH分别为70℃和8.0，在pH7.0-9.0范围内，具有较高的酶活性；该酶的K_m和K_(cat)/K_m分别为2.7mg mL~(-1)和51mL mg~(-1)s~(-1)，在80℃温度下处理30min，有60%的残余酶活；与解淀粉芽孢杆菌β-葡聚糖酶相比，热纤梭菌缺失锚定结构域β-葡聚糖酶的催化活性较低，但具有较高的热稳定性。
3.融合β-葡聚糖酶的构建。将热纤梭菌β-葡聚糖酶N-端13个和27个氨基酸残基分别与解淀粉芽孢杆菌β-葡聚糖酶N端融合，构建融合β-葡聚糖酶，分别命名为R13和R27。纯化后R13和R27的比活力分别为1074U mg~(-1)和1013U mg~(-1)，它们的最适反应温度和pH分别为60℃和6.0；在80℃温度下处理30min，R13和R27分别保留34%和52%的残余酶活，分别高出解淀粉芽孢杆菌β-葡聚糖酶残余酶活的18%和36%，但它们的耐热性均低于热纤梭菌β-葡聚糖酶。表明融合热纤梭菌β-葡聚糖酶N-端13个和27个氨基酸残基，能够提高解淀粉芽孢杆菌β-葡聚糖酶的耐热性。酶催化动力学研究表明，R13的K_m和K_(cat)/K_m分别为1.7mg mL~(-1)和324mL mg~(-1)s~(-1)，R27的K_m和K_(cat)/K_m分别为1.9mg mL~(-1)和288mL mg~(-1)s~(-1)。与解淀粉芽孢杆菌β-葡聚糖酶的催化活性相比较，R13和R27均有不同程度的降低，说明解淀粉芽孢杆菌N-端融合热纤梭菌β-葡聚糖酶N-端13或27个氨基酸残基后，降低了解淀粉芽孢杆菌β-葡聚糖酶与底物的亲和力，进而影响了酶的催化活性，其中融合热纤梭菌β-葡聚糖酶N-端27个氨基酸残基对解淀粉芽孢杆菌β-葡聚糖酶催化活性的抑制作用大于融合13个氨基酸残基的抑制作用。
4.双催化结构域β-葡聚糖酶的构建。将解淀粉芽孢杆菌β-葡聚糖酶的催化结构域和热纤梭菌β-葡聚糖酶的催化结构域连接，构建双催化结构域β-葡聚糖酶。构建的双催化结构域β-葡聚糖酶命名为RQ，纯化后RQ的比活力为2434U mg~(-1)，RQ的最适反应温度和pH分别为70℃和6.0；耐热性研究表明，在80℃温度下处理30min，RQ有67%的残余酶活，分别高出R13和R27残余酶活的33%和15%，RQ的耐热性与热纤梭菌β-葡聚糖的耐热性相接近；RQ的K_m和K_(cat)/K_m分别为1.2mg mL~(-1)和1014mL mg~(-1)s~(-1)，其催化效率分别是R13和R27催化效率的3.13倍和3.52倍，也是解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶催化效率之和的2.41倍。以上结果表明：RQ的酶学性质并不是简单的解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶的酶学性质的叠加，而是赋予了新的酶学特性的β-葡聚糖酶，它除具有热纤梭菌β-葡聚糖酶的热稳定性外，其催化效率也高于解淀粉芽孢杆菌β-葡聚糖酶和热纤梭菌β-葡聚糖酶的催化效率之和。热纤梭菌β-葡聚糖酶的连接子可有效改善双催化结构域β-葡聚糖酶的耐热性和催化活性。
5.双催化结构域β-葡聚糖酶的耐热机理初步研究。将RQ中热纤梭菌β-葡聚糖酶的两个催化活性位点的谷氨酸(E134和E138)定点突变为丙氨酸(E134A和E138A)，构建含点突变的双催化结构域β-葡聚糖酶，并命名为RQM。耐热性研究表明，在80℃温度下处理30min，RQM有57%的残余酶活，分别比解淀粉芽孢杆菌β-葡聚糖酶、R13和R27的耐热性提高了41%、23%和5%；与热纤梭菌β-葡聚糖酶和RQ的耐热性相比，RQM的耐热性分别降低了3%和10%。说明热纤梭菌β-葡聚糖酶不仅其N-端对RQ的耐热性有影响，而且其催化结构域的整个结构在稳定酶分子中发挥作用，包括其催化活性位点，热纤梭菌β-葡聚糖酶催化结构域和解淀粉芽孢杆菌β-葡聚糖酶催化结构域之间相互影响，产生协同效应，形成一种特性优良的、新的β-葡聚糖酶。
6.双催化结构域β-葡聚糖酶发酵培养基和培养条件如下：大麦粉43.48g L~(-1)、豆粉34.40g L~(-1)、NaCl2.40g L~(-1)、KH_2PO_42.40g L~(-1)和K_2HPO_412.50g L~(-1)；接种量1%(V/V)、装样量45mL/250mL、发酵培养基初始pH6.0-7.0、最佳诱导时期为在200rpm转速下培养3h后诱导。优化后的发酵培养基产β-葡聚糖酶的量为110U mL~(-1)，比初始发酵培养基提高了11%。虽然发酵产β-葡聚糖酶的量没有得到大幅度提高，但是优化后的发酵培养基中的碳源和氮源是廉价的大麦粉和豆粉，大大降低了β-葡聚糖酶的生产成本，具有很好的实际应用价值。
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~(-1)after 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~(-1)and369mL mg~(-1)s~(-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~(-1)after 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~(-1)and51mL mg~(-1)s~(-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~(-1)and1013U mg~(-1)after 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~(-1)s~(-1), respectively, while K_mand K_(cat)/K_mof R27were1.9mg mL~(-1)and288mL mg~(-1)s~(-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~(-1)afeter 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~(-1)and1014mL mg~(-1)s~(-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, KH_2PO_42.40and K_2HPO_412.50. 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.
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