β-甘露聚糖酶AuMan5A/Af酶学性质的改善与其Asp320的相关性分析

【目的】为改善宇佐美曲霉5家族β-甘露聚糖酶(AuMan5A)的酶学性质,本实验室前期将AuMan5A底物结合凹槽内一个7肽(~(316)KSPDGGN~(322))组成的loop替换为烟曲霉5家族β-甘露聚糖酶对应的氨基酸片段(PSPNDHF),得到loop替换突变酶AuMan5A/Af。为揭示AuMan5A/Af酶学性质显著改善与其Asp~(320)的相关性,定点突变构建突变体AuMan5A/Af~(D320G)。【方法】采用大引物PCR技术将AuMan5A/Af基因(Auman5A/Af)中编码Asp~(320)的密码子GAC突变为Gly~(320)的GGT,构建出突变体基因Auman5A/Af~(D320G),并在毕赤酵母GS115中进行表达,分析表达产物AuMan5A/Af~(D320G)的酶学性质。【结果】AuMan5A/Af~(D320G)的最适温度T_(opt)为70.0℃,变性温度T_m为71.5℃,介于AuMan5A(T_(opt)=65.0℃,T_m=64.5℃)和AuMan5A/Af(T_(opt)=75.0℃,T_m=76.6℃)之间;在70.0℃的半衰期为40 min,高于AuMan5A的10 min,但较AuMan5A/Af的480 min显著缩短;比活性分别是AuMan5A和AuMan5A/Af的2.7和0.3倍;催化效率(k_(cat)/K_m)分别是AuMan5A和AuMan5A/Af的3.9和0.3倍。【结论】将Asp~(320)突变为Gly~(320)显著影响了AuMan5A/Af的酶学性质,证明了Asp~(320)对AuMan5A/Af温度特性改善、比活性和催化效率显著提高的重要作用。     

定点突变提高苯丙氨酸羟化酶的热稳定性

嗜热菌中,蛋白质存在Ala替换Gly以及Arg替换Lys的趋势。为了提高紫色色杆菌来源的苯丙氨酸羟化酶的热稳定性,将该酶中所有Gly突变成Ala,Lys突变成Arg,筛选获得热稳定性提高的突变体,并进行组合突变,对突变酶的酶学性质进行研究。结果表明,突变酶K94R和G221A在50℃的半衰期分别为26.2 min、16.8 min,比原始酶(9.0 min)分别提高了1.9倍、0.9倍,同时组合突变酶K94R/G221A在50℃处理1 h后仍保留65.6%的酶活,比原始酶(8.6%)高出6.6倍。圆二色谱结果显示原始酶和突变酶K94R、G221A及K94R/G221A的T_m值分别为51.5℃、53.8℃、53.1℃和54.8℃。蛋白三维结构模拟推测突变体热稳定性提高机理为:突变体K94R中Arg94与Ile95之间形成额外氢键,稳定其所在的柔性区域;突变体G221A中Ala221与Leu281产生疏水作用,稳定酶分子C-端柔性区。该研究结果为蛋白质热稳定性改造提供了参考,也为苯丙氨酸羟化酶在功能性食品领域的应用奠定了基础。     

β-甘露聚糖酶末端残基变体对其酶学特性的影响

本研究用宇佐美曲霉Aspergillus usamii的5家族β-甘露聚糖酶AuMan5A为母本,借助同源建模、分子对接及分子动力学模拟等理性设计方法,将AuMan5A的N-末端和C-末端分别截去3个无规则的氨基酸残基,构建出截短的β-甘露聚糖酶 AuMan5A^N3C3.将AuMan5A和AuMan5A^N3C3的编码基因分别在毕赤酵母GS115中进行表达,对表达产物进行了初步纯化并分析比较了其酶学特性及各自的表达水平. 结果表明,reAuMan5A和reAuMan5A^N3C3的最适温度Topt均为70 ℃,reAuMan5A^N3C3在60 ℃的半衰期t_1/2⁶⁰为38 min,较reAuMan5A（t_1/2⁶⁰=40 min）略有降低;在相同表达条件下,reAuMan5A^N3C3上清液的β-甘露聚糖酶活性为73.4 U/mL,较reAuMan5A 的52.8 U/mL提高了39.0%;纯化的reAuMan5A^N3C3酶比活性为182.7 U/mg protein,较reAuMan5A的126.3 U/mg protein提高了44.7%. 与reAuMan5A相比,reAuMan5A^N3C3对角豆胶的Km值下降不明显,Vmax值有显著提高.     

N端二硫键对11家族木聚糖酶热稳定性的影响

以来源于米曲霉Aspergillus oryzae的11家族常温木聚糖酶AoXyn11A为母本,将其N端替换成同一家族耐热木聚糖酶EvXyn11TS的对应片段,构建出耐热杂合木聚糖酶AEx11A。将AoXyn11A和AEx11A基因分别在毕赤酵母GS115中进行表达并分析比较温度对表达产物酶活性的影响。结果表明,AEx11A的最适温度Topt为75℃,在70℃的半衰期t1/270为197 min,较AoXyn11A(Topt=50℃,t1/270=1.0 min)显著提高。通过对AEx11A结构的同源建模及其与AoXyn11A结构的比对,发现在AEx11A的N端引入了一个二硫键(Cys5–Cys32)。利用定点突变法将其5位的半胱氨酸突变为苏氨酸(C5T),去除该二硫键,以探讨其对AEx11A热稳定性的影响。分析表明,突变酶(AEx11AC5T)的Topt由突变前的75℃降为60℃,其t1/270和t1/280也分别由197 min和25 min缩短为3.0 min和1.0 min。     

天蓝色链霉菌海藻糖合酶的异源表达、活性分析及重组菌全细胞转化合成海藻糖的条件优化

从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因 (ScTreS),在大肠杆菌Escherichia coli BL21(DE3) 中进行了异源表达,通过 Ni-NTA 亲和柱对表达产物进行分离纯化得到纯酶,经 SDS-PAGE 测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35 ℃;最适pH 7.0,对酸性条件比较敏感。通过同源建模和序列比对分析,对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍,突变酶A165T相对提高了1.39倍,海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5 L罐发酵,结果表明：在麦芽糖浓度300 g/L、初始反应温度和pH分别为35 ℃和7.0的条件下,转化率最高达到71.3%,产量为213.93 g/L;当底物浓度增加到700 g/L时,海藻糖产量仍可达到465.98 g/L。     

置换β-甘露聚糖酶底物结合槽内环结构序列改善其酶学特性

位于酶分子活性位点(又称活性中心)附近的环结构对酶促反应特性具有重要影响.本实验室先前在宇佐美曲霉Aspergillus usamii中发现了一种新的5家族β-甘露聚糖酶Au Man5A.通过同源建模、分子对接及多序列比对,发现位于Au Man5A底物结合凹槽侧壁的一段环结构可能对酶的功能有影响.为证明该假说,本研究采用Au Man5A为母本,利用PCR技术将其底物结合槽内的7肽环结构316KSPDGGN322替换成与两种木霉属β-甘露聚糖酶对应的8肽片段AQSNSDPY,构建了杂合酶基因Auman5ALoop,并在毕赤酵母GS115中进行表达,经纯化获得了杂合β-甘露聚糖酶Au Man5ALoop.酶学特性分析结果显示,与原酶Au Man5A比较,Au Man5ALoop的最适反应p H由3.5~4.0扩宽至3.5~5.5;最适反应温度由65℃提高至70℃;以角豆胶为底物,测得Au Man5ALoop的比活性由351.2 U/mg提高至2 089.2 U/mg.利用双倒数作图法测得动力学常数揭示,Au Man5ALoop的Km值较Au Man5A降低了36%,而kcat值是Au Man5A的6.8倍;同时,Au Man5ALoop的催化效率(kcat/Km)提高了10.7倍.我们的结果说明,通过替换Au Man5A底物结合槽内环结构,可明显改进其酶学特性.我们的结果还提示,活性位点附近的环结构对β-甘露聚糖酶的酶学特性具有重要影响.     

V1C、G116D、N171H定点突变对黑曲霉(Aspergillus niger)XZ-3S木聚糖酶XynZF-2热稳定性的影响

[目的]探索V1C、G116D、N171H定点突变对木聚糖酶Xyn ZF-2热稳定性的作用。[方法]生物信息学方法确定木聚糖酶Xyn ZF-2可突变位点,引入Cys、Asp、His;定点突变V1C、G116D、N171H,PCR扩增突变基因xyn CDH,构建大肠杆菌表达载体,转化表达宿主大肠杆菌BL21(DE3),诱导表达目的蛋白并测定酶活,分析酶学性质。[结果]突变酶Xyn CDH的最适温度由40℃上升到45℃。40℃条件下突变酶Xyn CDH半衰期t40℃1/2由55 min延长到60 min;在45℃条件下,突变酶Xyn CDH的半衰期t45℃1/2由7min提高到15 min。[结论]定点突变V1C、G116D、N171H能增强木聚糖酶Xyn ZF-2热稳定性,为加深木聚糖酶分子改造的研究提供参考。     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

普鲁兰酶基因在毕赤酵母中组成型表达及定点突变研究

合成Bacillus acidopullulyticus的全长普鲁兰酶基因并在毕赤酵母X-33中进行组成型外分泌表达,重组酶的最适作用温度为60℃,最适作用pH值为4.5~5.0,酶比活力为2.0 U/mg.采用重叠延伸PCR方法对普鲁兰酶基因进行定点突变,实验结果表明,625、626位点Ala、Leu氨基酸突变为Leu、Tyr氨基酸后,该酶的催化效率有所降低,而Gln487Ala的突变对催化效率没有较大的影响.该研究结果为探究关键氨基酸区域对催化效率的影响提供了一定的理论和实验基础.     

K178M、S170F、G180V突变对木聚糖酶XynZF-2热稳定性的影响

利用生物信息学分析黑曲霉木聚糖酶Xyn ZF-2,选择α-螺旋178位点、170位点和180位点氨基酸进行定点突变(K178M,S170F,G180V)获得突变木聚糖酶基因xyn MFV,构建重组表达载体转化大肠杆菌E.coli BL21(DE3)诱导表达。酶学性质分析对比发现,突变酶最适温度(50℃)比原酶(40℃)提高了10℃;40℃条件下保温1 h突变酶Xyn MFV相对酶活力下降到热处理前的56.0%,原酶Xyn ZF-2下降到42.0%;45℃时突变酶Xyn MFV的半衰期t_(1/2)为21 min,与原酶Xyn ZF-2(t_(1/2)=7 min)相比较提高了14 min。结果表明,K178M、S170F、G180V突变木聚糖酶可以提高Xyn ZF-2最适温度和热稳定性。     

低致死剂量氯虫苯甲酰胺对沟金针虫食物利用和相关生理生化指标的剂量和时间效应

【目的】明确氯虫苯甲酰胺对沟金针虫Pleonomus canaliculatus亚致死效应的生理生化机制,阐明氯虫苯甲酰胺低致死剂量对沟金针虫食物利用、能量物质含量以及体内消化酶、保护酶和解毒酶活力的影响。【方法】室内采用土壤混药法测定氯虫苯甲酰胺对沟金针虫3龄幼虫毒力,并测定了氯虫苯甲酰胺LC₁₀, LC₂₅和LC₄₀低致死剂量对沟金针虫3龄幼虫营养指标和体内能量物质含量的影响;采用酶动力学法检测了氯虫苯甲酰胺低致死剂量处理1, 6, 12, 24, 48和72 h后沟金针虫3龄幼虫体内消化酶(蛋白酶、α-淀粉酶、脂肪酶、海藻糖酶)、保护酶(CAT, POD和SOD)以及解毒酶(CarE, MFO和GST)活力的动态变化。【结果】氯虫苯甲酰胺对沟金针虫3龄幼虫有较高毒力,其LC₅₀值为1.2397 mg/kg。LC₁₀和LC₄₀剂量氯虫苯甲酰胺处理沟金针虫3龄幼虫后,平均相对生长率(MRGR)和近似消化率(AD)显著降低,严重干扰其对食物的利用;LC₁₀, LC₂₅和LC₄₀剂量处理后沟金针虫3龄幼虫体内主要的能量物质(蛋白质、脂质、碳水化合物、海藻糖)含量和消化酶活力均明显降低,而解毒酶和保护酶活力显著增加,最终延缓其生长发育。【结论】氯虫苯甲酰胺对沟金针虫幼虫具有很高的杀虫活性,低致死剂量氯虫苯甲酰胺处理沟金针虫幼虫后,通过抑制消化酶活性,使其对食物的利用能力降低和生长发育延缓,以及诱导解毒酶和保护酶活性来阻止外界毒物侵害。研究结果为阐明氯虫苯甲酰胺对沟金针虫的亚致死效应机制及作用机理提供了一定的理论基础。     

梨小食心虫幼虫中肠中高表达消化酶和解毒酶基因的筛选

【目的】挖掘梨小食心虫Grapholita molesta幼虫中肠中高表达消化酶和解毒酶基因,为今后研究以肠道为靶标的新型农药和转基因作物提供理论依据。【方法】基于梨小食心虫4龄幼虫中肠转录组高通量测序数据的FPKM值,筛选高表达基因,进行GO功能注释和KEGG通路富集分析,并使用BLAST软件进行比对筛选高表达的消化酶和解毒酶基因,利用MEGA对这些高表达的消化酶和解毒酶及其他鳞翅目昆虫的同源蛋白进行系统发育分析。利用qRT-PCR技术对梨小食心虫幼虫不同龄期中肠中的高表达代表性消化酶和解毒酶基因表达量进行定量分析和验证。【结果】在GO数据库中注释了103 677个在梨小食心虫4龄幼虫中肠中高表达基因,包括细胞组分、分子功能和生物学进程三大类功能共41个分支。KEGG通路分析表明,10 846个高表达基因参与了5类生化代谢通路。筛选到具有完整开放阅读框的消化酶基因17个[5个胰蛋白酶(trypsin, TRY)基因、3个氨肽酶(aminopeptidase, APN)基因和9个羧肽酶(carboxypeptidase, CP)基因]和解毒酶基因32个[11个谷胱甘肽S-转移酶(glu...     

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn²⁺ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M²⁺) cofactors, except Ca²⁺, for catalysis. We found that Zn²⁺ yielded the highest enzyme complex thermostability (T_m of 87 °C) and solvent tolerance. All AbHpaI•M²⁺ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn²⁺ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M²⁺ cofactors). For the aldol condensation reaction, AbHpaI•M²⁺ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M²⁺ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M²⁺ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn²⁺-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca²⁺ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn²⁺ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.     

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS*

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL^pro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL^pro from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL^pro is less efficient at processing a peptide substrate due to MERS-CoV 3CL^pro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL^pro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL^pro is a weakly associated dimer (K_d ∼52 μm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL^pro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL^pro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL^pro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL^pro dimerization. Activation of MERS-CoV 3CL^pro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL^pro inhibitors as antiviral agents.     

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis

Cytochrome P450 (P450) 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the subsequent lyase cleavage of both products to generate androgens. However, the selective inhibition of the lyase reactions, particularly with 17α-hydroxy pregnenolone, remains a challenge for the treatment of prostate cancer. Here, we considered the mechanisms of inhibition of drugs that have been developed to inhibit P450 17A1, including ketoconazole, seviteronel, orteronel, and abiraterone, the only approved inhibitor used for prostate cancer therapy, as well as clotrimazole, known to inhibit P450 17A1. All five compounds bound to P450 17A1 in a multistep process, as observed spectrally, over a period of 10 to 30 s. However, no lags were observed for the onset of inhibition in rapid-quench experiments with any of these five compounds. Furthermore, the addition of substrate to inhibitor–P450 17A1 complexes led to an immediate formation of product, without a lag that could be attributed to conformational changes. Although abiraterone has been previously described as showing slow-onset inhibition (t_1/2 = 30 min), we observed rapid and strong inhibition. These results are in contrast to inhibitors of P450 3A4, an enzyme with a larger active site in which complete inhibition is not observed with ketoconazole and clotrimazole until the changes are completed. Overall, our results indicate that both P450 17A1 reactions—17α-hydroxylation and lyase activity—are inhibited by the initial binding of any of these inhibitors, even though subsequent conformational changes occur.     

High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain–containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80–2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1–2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.     

Molecular mechanism of intramolecular electron transfer in dimeric sulfite oxidase

Sulfite oxidase (SOX) is a homodimeric molybdoheme enzyme that oxidizes sulfite to sulfate at the molybdenum center. Following substrate oxidation, molybdenum is reduced and subsequently regenerated by two sequential electron transfers (ETs) via heme to cytochrome c. SOX harbors both metals in spatially separated domains within each subunit, suggesting that domain movement is necessary to allow intramolecular ET. To address whether one subunit in a SOX dimer is sufficient for catalysis, we produced heterodimeric SOX variants with abolished sulfite oxidation by replacing the molybdenum-coordinating and essential cysteine in the active site. To further elucidate whether electrons can bifurcate between subunits, we truncated one or both subunits by deleting the heme domain. We generated three SOX heterodimers: (i) SOX/Mo with two active molybdenum centers but one deleted heme domain, (ii) SOX/Mo_C264S with one unmodified and one inactive subunit, and (iii) SOX_C264S/Mo harboring a functional molybdenum center on one subunit and a heme domain on the other subunit. Steady-state kinetics showed 50% SOX activity for the SOX/Mo and SOX/Mo_C264S heterodimers, whereas SOX_C264S/Mo activity was reduced by two orders of magnitude. Rapid reaction kinetics monitoring revealed comparable ET rates in SOX/Mo, SOX/Mo_C264S, and SOX/SOX, whereas in SOX_C264S/Mo, ET was strongly compromised. We also combined a functional SOX Mo domain with an inactive full-length SOX R217W variant and demonstrated interdimer ET that resembled SOX_C264S/Mo activity. Collectively, our results indicate that one functional subunit in SOX is sufficient for catalysis and that electrons derived from either Mo^(IV) or Mo^(V) follow this path.     

A hierarchical deep learning based approach for multi‐functional enzyme classification

Enzymes are critical proteins in every organism. They speed up essential chemical reactions, help fight diseases, and have a wide use in the pharmaceutical and manufacturing industries. Wet lab experiments to figure out an enzyme''s function are time consuming and expensive. Therefore, the need for computational approaches to address this problem are becoming necessary. Usually, an enzyme is extremely specific in performing its function. However, there exist enzymes that can perform multiple functions. A multi‐functional enzyme has vast potential as it reduces the need to discover/use different enzymes for different functions. We propose an approach to predict a multi‐functional enzyme''s function up to the most specific fourth level of the hierarchy of the Enzyme Commission (EC) number. Previous studies can only predict the function of the enzyme till level 1. Using a dataset of 2,583 multi‐functional enzymes, we achieved a hierarchical subset accuracy of 71.4% and a Macro F₁ Score of 96.1% at the fourth level. The robustness of the network was further tested on a multi‐functional isoforms dataset. Our method is broadly applicable and may be used to discover better enzymes. The web‐server can be freely accessed at http://hecnet.cbrlab.org/.     

长足大竹象内切葡聚糖酶最适反应条件的确定及其关键基因的筛选

【目的】本研究旨在阐明长足大竹象Cyrtotrachelus buqueti内切葡聚糖酶最适反应条件,并挖掘长足大竹象消化道内切葡聚糖酶关键基因。【方法】采用3,5-二硝基水杨酸(DNS)法,设置以羧甲基纤维素钠(MC)为反应底物的单因素和正交优化试验测定内切葡聚糖酶反应的最适条件。通过对长足大竹象发育转录组内切葡聚糖酶编码基因进行生物信息学分析,并将基因表达量与酶活性数据进行关联分析,筛选出发育时期中关键内切葡聚糖酶基因,采用实时荧光定量PCR对不同发育时期长足大竹象消化道内切葡聚糖酶关键基因表达量进行验证确定。【结果】研究表明,长足大竹象成虫内切葡聚糖酶的最适反应条件为：温度45℃,pH 5.6,底物浓度2%,酶比活力59.85 U/mg(雌)和52.87 U/mg(雄);幼虫内切葡聚糖酶的最适反应条件为：温度35℃,pH 4.8,底物浓度2％,酶比活力38.34 U/mg。筛选出长足大竹象消化道内切葡聚糖酶关键基因c64192_g1和c57057_g1。实时荧光定量PCR结果表明c64192_g1和c57507_g1基因在成虫时期表达量高于幼虫。【结论】长足大竹象雌雄成虫的内切葡聚糖酶比活力均高于幼虫,存在两个影响内切葡聚糖酶活性的关键基因c64192_g1和c57507_g1。这些研究成果丰富了内切葡聚糖酶来源,并为长足大竹象内切葡聚糖酶异源表达提供数据参考,进而为木质纤维素预处理和生物质能源的开发利用奠定理论基础。