新型GH5家族多结构域纤维素酶的结构与功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0641

摘要/Abstract

摘要：

纤维素酶能够将纤维素转化为可发酵的糖类，为丰富纤维素酶的序列与结构资源、揭示纤维素酶结构与功能之间的关系，本研究对两个新型GH5家族多结构域内切纤维素酶TlCel5和ReCel5进行了克隆表达和酶学性质测定，并对其结构域开展了突变研究。序列和结构分析显示，Tlcel5和Recel5分别编码了655个和632个氨基酸，理论分子量分别为68.3 kD和65.9 kD，均包含了CBM1区、CD区、CBMX2区和一个未知结构域，这与以往报道的多数单一结构域或双结构域纤维素酶显著不同。为了解附加结构域对酶功能的影响效果，以ReCel5为研究对象，分别构建了N端CBM1结构域的截断突变体TM1，和C端未知结构域的截断突变体TM2。酶学性质分析显示，TlCel5和ReCel5的最适作用pH和最适作用温度分别为pH 3.0、50℃和pH 4.0、70℃，在50℃和70℃下能够保持良好的稳定性，并且对多种类型的纤维素类底物、半纤维素类底物表现出水解能力。虽然突变体TM1和TM2的酶学性质较野生型没有发生显著变化，但其对羧甲基纤维素钠、大麦葡聚糖、地衣多糖的水解比活值降低了23%-68%，由此说明，在多结构域酶中，附加结构域与酶的水解能力之间存在密切关系。

关键词: 多结构域内切纤维素酶, 克隆表达, 酶学性质, 结构与功能

Abstract:

Cellulase can convert cellulose into fermentable sugars. In order to enrich the sequences and structure resources of cellulase and reveal the relationship between cellulase structure and function, two novel multi-domain endoglucanases belonging to glycosyl hydrolases（GH）family 5 TlCel5 and ReCel5 were cloned and expressed. Their enzymatic properties were determined and the mutation of structure domain was studied. Sequence and structure analysis showed that Tlcel5 and Recel5 encoded 655-residue and 632-residue polypeptides with theoretical molecular weights of 68.3 kD and 65.9 kD respectively. They wee both multi-domain cellulases, comprising a carbohydrate-binding module grouped into family 1（CBM1）and a catalytic module of family 5 glycoside hydrolase（CD）at N-terminal, a family X2 carbohydrate binding module（CBMX2）and an unknown domain（UM）at the C-terminal, which made them significantly different from most single-domain or dual-domain cellulases reported previously. With the purpose of understanding the effect of additional domains on enzyme function, the truncated mutant without the N-terminal CBM1 domain（TM1）and the truncated mutant without the C-terminal unknown domain（TM2）were constructed based on wild-type ReCel5. The assays of enzymatic properties demonstrated that the optimal pH and temperature of TlCel5 and ReCel5 were pH 3.0, 50℃ and pH 4.0, 70℃, respectively. They remained fine stability at 50℃ and 70℃, and showed certain hydrolytic ability to a variety of cellulose and hemicellulose substrates. Their hydrolysis specific viabilities to sodium carboxymethyl cellulose, barley β-glucan, and lichenan reduced by 23% to 68% although the enzymatic properties of TM1 and TM2 did not change significantly compared with the wild-type proteins. These results suggested that there was a close relationship between the additional domains and the hydrolysis ability of multi-domain enzymes.

Key words: multi-domain endoglucanase, cloning expression, enzymatic property, structure and function

杨俊钊, 张新蕊, 赵国柱, 郑菲. 新型GH5家族多结构域纤维素酶的结构与功能研究[J]. 生物技术通报, 2023, 39(4): 71-80.

YANG Jun-zhao, ZHANG Xin-rui, ZHAO Guo-zhu, ZHENG Fei. Structure and Function Analysis of Novel GH5 Multi-domain Cellulase[J]. Biotechnology Bulletin, 2023, 39(4): 71-80.

图/表 8

图1 TlCel5、ReCel5、TM1和TM2编码蛋白质结构示意图 SP：信号肽；CBM：碳水化合物结合模块；CD：催化结构域；UM：未知结构域

Fig. 1 Schematic diagram of protein structure encoded by TlCel5, ReCel5, TM1 and TM2 SP: Signal peptide. CBM: Carbohydrate binding module. CD: Catalysis domain. UM: Unknown module

图2 GH5家族内切纤维素酶催化结构域氨基酸序列对比图保守残基由向下箭头所标记。比对序列的菌株来源及登录号分别为：H. jecorina（AEJ36301.1）, R. emersonii CBS 393.64（XP_013323622.1）, Aspergillus neoniger CBS 115656（XP_025478804.1）, Thermoascus aurantiacus（AAL88714.2）, Stegonsporium opalus（ARO48344.1）, Ganoderma lucidum（QDK64599.1）, Trichoderma reesei（P07982.1）, and Gloeophyllum trabeum ATCC 11539（XP_007867902.1）

Fig. 2 Amino acids sequence alignment in the catalytic domain of GH5 family endoglucanases The conservative residues are labeled in the arrow below the alignment. The strain source and GenBank number of the alignment sequence are H. jecorina（AEJ36301.1）, R. emersonii CBS 393.64（XP_013323622.1）, A. neoniger CBS 115656（XP_025478804.1）, T. aurantiacus（AAL88714.2）, S. opalus（ARO48344.1）, G. lucidum（QDK64599.1）, T. reesei（P07982.1）, and G. trabeum ATCC 11539（XP_007867902.1）

图3 野生型TlCel5、ReCel5和突变体TM1、TM2的SDS-PAGE分析 M：蛋白分子质量标准； A：野生型TlCel5、ReCel5的表达（1：ReCel5纯化蛋白；2：TlCel5纯化蛋白）； B：突变体TM1、TM2的表达（3：突变体TM1纯化蛋白；4：突变体TM2纯化蛋白）

Fig. 3 SDS-PAGE analysis of wild-type TlCel5, ReCel5 and mutant TM1, TM2 M: Protein molecular weight standard. A: Expression of wile-type TlCel5、ReCel5（1: purified protein of ReCel5; 2: purified protein of TlCel5）. B: Expressions of mutants TM1 and TM2（3: purified protein of TM1; 4: purified protein of TM2）

图4 野生型TlCel5、ReCel5及突变体TM1、TM2的酶学性质图 A：最适pH；B：pH稳定性；C：最适温度；D：50℃下温度稳定性；E：60℃下温度稳定性；F：70℃下温度稳定性

Fig. 4 Enzymatic properties of wild-type TlCel5, ReCel5 and mutant TM1, TM2 A: pH-activity profile. B: pH stability. C: Temperature-activity profile. D: Thermostability at 50℃. E: Thermostability at 60℃. F: Thermostability at 70℃

图5 野生型TlCel5、ReCel5及突变体TM1、TM2的比活值

Fig. 5 Specific viability of wild-type TlCel5, ReCel5 and mutant TM1, TM2

表1 野生型TlCel5、ReCel5及突变体TM1、TM2的酶促反应动力学参数

Table 1 Kinetics parameters of wild-type TlCel5, ReCel5 and mutants TM1, TM2

Enzyme	V_max/（mmol·min^-1·mg^-1）	K_m/（mg·mL^-1）	k_cat/（s^-1）	k_cat/K_m（mL·mg^-1·s^-1）
TlCel5	217.0±20.0	7.5±1.0	247.0±22.0	32.8±1.5
ReCel5	56.0±0.9	8.6±0.2	61.4±0.9	7.1±0.1
TM1	68.0±0.5	8.4±0.1	70.7±0.6	8.4±0.1
TM2	76.9±0.7	8.4±0.1	68.1±0.6	8.1±0.1

表2 GH5家族真菌源内切纤维素酶最适条件表

Table 2 Optimal conditions for endoglucanases of GH5 family fungi

Name	Optimal pH	Optimal temperature /℃	Source	Reference
TlCel5	3.0	50	T. leycettanus	This study
ReCel5	4.0	70	R. emersonii	This study
GlCel5A	3.0-4.0	60	Ganoderma lucidum	[8]
TaCel5A	6.0	50	Thermoascus aurantiacusIFO9748	[9]
Epi3	6.5-7.0	50	Epidinium caudatum	[10]
PdCel5C	4.8	40-50	Penicillium decumbens 114-2	[11]
BaCel5	5.0	50	Bispora antennata	[12]
SoCel5	5.0	60	Stegonsporium opalus	[13]

图6 纤维素酶CBM1序列比对比对序列的纤维素酶名称、来源菌株及登录号分别为：ReCel5（R.emersonii CBS 393.64, XP_013323622.1）, TrCel7A（T. reesei, CAH10320.1）, TrCel7B（T. reesei, AAA34212.1）, PdCel5C（P. decumbens, JQ319040.1）, TrCel5C（T. reesei, AAA34213.1）, ApCel5A（Aureobasidium pullulans, AEM23896.1）

Fig. 6 Sequence alignment of CBM1 domain of cellulose The cellulase name, source strain and GenBank number of the alignment sequence are ReCel5（R. emersonii CBS 393.64, XP_013323622.1）, TrCel7A（T. reesei, CAH10320.1）, TrCel7B（T. reesei, AAA34212.1）, PdCel5C（P. decumbens, JQ319040.1）, TrCel5C（T. reesei, AAA34213.1）, ApCel5A（Aureobasidium pullulans, AEM23896.1）

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