人双专一性磷酸酶活性位点Cys^124附近精氨酸突变及功能

为研究人双专一性磷酸酶活性位点Cys12 4 附近 3个带正电的精氨酸对酶催化功能的影响 ,用QuikChange定点突变方法获得 6个突变体 :R12 5L、R130 L、R130 K、R130 L/S131A、R158K和R158L。将含突变基因的重组质粒转化大肠杆菌菌株BL2 1(DE3) ,经IPTG诱导表达获得的目的蛋白质均以可溶形式存在。通过镍离子亲和层析纯化得到纯度大于 90 %的蛋白质。对人痘苗病毒H1相关磷酸酶 (VHR)及其突变体进行稳态动力学参数和竞争性抑制常数Ki 的测定 ,结果显示上述Arg130 和Arg158突变体的kcat/Km 值都较野生型有大幅度下降 ,而Ki 值有明显上升 ,表明 130和 15 8位的精氨酸是VHR活性所必需 ,而且可能与底物上带负电的磷酸基团结合有关。另外 ,单突变体R130 L和双突变体R130 L/S131A之间的kcat值相差很小 ,提示Arg130 单点突变后可能破坏了Ser131与Cys12 4 间的氢键。再者 ,R12 5L、R130 L和R158L突变体都降低了砷酸盐结合亲和性 ,暗示这 3个精氨酸残基侧链上的正电荷可能有助于底物与酶的结合。     

定向引入N-糖基化位点改进黑曲霉β-1,4-内切木聚糖酶热稳定性

【背景】木聚糖是生物圈中仅次于纤维素的第二大多糖,其结构复杂,完全降解需要多种木聚糖酶协同作用。β-1,4-内切木聚糖酶是木聚糖主链水解过程中最关键的酶,已广泛应用于饲料、造纸、能源、食品和医药等行业。但在实际应用中,由于真菌木聚糖酶的热稳定性较差,限制了其在工业中的应用。【目的】提高来源于黑曲霉(Aspergillusniger)的β-1,4-内切木聚糖酶(xynB)热稳定性。【方法】采用氨基酸虚拟突变技术对xynB定向引入一个N-糖基化位点,将虚拟突变后筛选获得的候选突变体和野生型在毕赤酵母SMD1168中表达,并对纯化后的野生型和突变体酶进行酶学性质和稳定性分析。【结果】经虚拟突变和筛选获得5个候选突变体,在毕赤酵母SMD1168中成功表达了4个突变体,其中3个突变体发生了糖基化。突变体和野生型酶均表现出宽范围的酸碱耐受性,且突变体xynB~(A92N/D94T)在pH4.0–11.0条件下的稳定性明显优于野生型;糖基化突变体xynB~(A92N/D94T)、xynB~(G66N/A68T)和xynB~(G66F/D67N/G69T)在温度为60–80°C时热稳定性明显高于野生型,xynB~(G66N/A68T)在80°C保温30 min后的残留酶活比野生型提高了约30%。【结论】本研究方法可为其他来源木聚糖酶和其他工业酶的热稳定分子改造提供参考。     

扩展青霉脂肪酶K56R叠加突变对热稳定性的影响

目的:扩展青霉脂肪酶随机突变体ep8是一株热稳定性比野生型有所提高的突变体.获得热稳定性提高的优良菌株.方法:在ep8的基础上利用重叠延伸PCR构建叠加突变重组质粒pPIC3.5K-ep8一K56R,将该质粒电转毕赤酵母(Pichia paaoris)GS115进行异源表达.结果:该叠加突变脂肪酶在毕赤酵母中获得了活性表达.15%SDS-PACE结果分析表明突变脂肪酶PEL-ep8-K56R-GS分子量与野生型PEL-GS一致,约为28kDa.叠加突变脂肪酶在37℃时酶活为852U/mL、野生型为760u/mL、随机突变体为824u/mL,叠加突变体酶活相比野生型提高了21.1%,相比随机突变体提高了3.4%.热稳定性分析数据表明叠加突变脂肪酶Tm值为40.1℃、野生型为38.7℃、随机突变体为39.9℃,Tm值相比野生型提高了1.4℃,相比随机突变体提高了0.2℃.     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达。实验结果表明:该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS(627u/mL)的81%,随机突变脂肪酶PEL-ep8-GS(924u/mL)的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其Tm值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

氨基酸序列突变对TFPI生物半衰期和生物活性的影响

通过对个别氨基酸突变的研究,获得了保持良好生物活性的长半衰期组织因子途径抑制因子(tissue factor pathwayinhibitor,TFPI)重组蛋白的有效途径.采用定点诱变和基因重组技术,首先在TFPI cDNA特定位点形成一个位点的沉默突变,以提高TFPI在毕赤酵母细胞内的表达量,此cDNA称为mTFPI.在此基础上,通过系列位点突变,形成3个羧基端突变体:m0TFPI、m1TFPI和m2TFPI.将上述4种TFPI cDNA与表达质粒pPic9连接,转染大肠杆菌,通过PCR和DNA测序确认重组质粒,转染酵母细胞GS115,甲醇诱导表达重组蛋白.采用层析方法纯化TFPI重组蛋白,用125I标记重组蛋白,静脉注射给药,比较四者在SD大鼠体内血浆代谢清除速度.用底物显色法测定重组蛋白抑制凝血因子Xa(Fxa)的活性,比较各株TFPI重组蛋白突变体在体内、体外对FXa的抑制作用及肝素对各株TFPI重组蛋白功能的影响.结果显示,相比野生型TFPI重组蛋(mTFPI)而言,3株羧基端突变体m0TFPI、m1TFPI、m2TFPI在SD大鼠体内血浆代谢清除时间均有不同程度延长,其生物代谢半衰期分别是mTFPI的1.5倍、1.9倍和大于2倍,与m-TFPI相比,3个rTFPI突变体在体内、体外抑制FXa的作用无明显减弱,与肝素的结合能力及协同能力也无明显减弱.结果表明,m0TFPI、m1TFPI和m2TFPI在生物半衰期得到明显延长的同时,仍保持良好的抑制Fxa的生物活性.     

黄翅大白蚁来源β-葡萄糖苷酶的分子改造

纤维素水解成为葡萄糖需要一系列纤维素酶的作用,其中β-葡萄糖苷酶(β-glucosidases)起着至关重要的作用。来自于培菌白蚁中肠的β-葡萄糖苷酶(MbmgBG1)具有较高的葡萄糖耐受性(1.5 mol/L的葡萄糖,保持60%以上的酶活力),但是,酶活力低和热稳定性差限制了β-葡萄糖苷酶(MbmgBG1)在食品以及工业领域中的应用。因此通过对保守氨基酸附近的非保守氨基酸定点突变,获得点突变体(F167L、T176C、E347I、R354K、N393G和V425M),其中突变体F167L、R354K的比活力(底物pNPG)比MbmgBG1分别高出约2倍和4倍。突变体的K_(cat)/K_m值比野生型大,反映了突变体对底物的亲和力以及催化能力比MbmgBG1强。当酶活力保留60%以上时,MbmgBG1所耐受的葡萄糖浓度为1.5 mol/L,而F167L为2.0 mol/L,R354K为3.0 mol/L。这些特性的增强表明,对活性中心附近保守区域内的非保守氨基酸突变,可以较大程度地影响活性,因此需要更深入地研究β-葡萄糖苷酶的活性中心位点,进行改造以提高催化效率。     

人白细胞介素18突变体的构建及其功能

为研究人白细胞介素 18(hIL 18)结构与功能的关系 ,用PCR定点突变技术分别构建了N末端、C末端缺失突变体(ΔNC)和IL 1特征样序列突变体S154A/Y156F/E157P/C163 T (S)。将突变体cDNA与原核表达载体 pJW 2重组并转化大肠杆菌DH5α ,经热诱导表达蛋白质 ,SDS PAGE证实表达的目的蛋白质以包涵体形式存在。菌体经超声破碎后 ,包涵体以 2mol/L尿素洗涤 ,8mol/L尿素溶解 ,并经SephadexG 75柱纯化 ,纯度可达 95 %以上。突变体蛋白质经逐步稀释复性后 ,以诱导人外周血单个核细胞 (PBMC)产生干扰素 γ(IFN γ)及对核因子 κB(NF κB)的激活能力为指标 ,检测突变体的生物学活性。结果显示ΔNC、S这 2个突变体对IFN γ的诱生能力显著低于野生型hIL 18,分别为野生型hIL 18的 13%和 4 8%。同时 ,ΔNC、S对NF κB的激活能力也低于野生型hIL 18,分别为野生型hIL 18的 6 9.7%和 89.8%。这些结果表明缺失的片段或突变的位点对hIL 18的功能有重要的作用。     

人博卡病毒2型VP1U突变对sPLA2活性的影响

对HBoV2 VP1U蛋白进行原核表达并检测其sPLA_2活性,对其关键氨基酸进行突变,检测突变对sPLA_2活性的影响。构建重组质粒pMD18T-VP1U,选取VP1U蛋白4个关键氨基酸为突变点,分别进行原核表达,蛋白纯化后用sPLA_2试剂盒检测sPLA_2活性,对比突变前后VP1U蛋白sPLA_2活性有无变化。野生型HBoV2VP1U蛋白的sPLA_2活性为0.243μmol/min/mL,具有sPLA_2活性;突变体L19P、P21R、D42H及Y45N蛋白的sPLA_2活性分别为野生型蛋白的1.2%、1.2%、0.82%、0.41%。野生型HBoV2VP1U蛋白具有sPLA_2活性,突变体蛋白均几乎完全丧失了sPLA_2活性,提示19、21、42、45位4个氨基酸是维持sPLA_2活性的关键氨基酸。该研究为靶向抗病毒药物的研究提供了理论基础。     

鸡α干扰素在毕赤酵母中的表达及糖基化对其表达的影响

目的:实现鸡α干扰素(ChIFNα)在毕赤酵母GS115中的表达,并考察糖基化对其表达的影响。方法:人工合成按照毕赤酵母偏好密码子优化的ChIFNα基因,克隆至分泌表达载体pPIC9,电转到毕赤酵母GS115中进行表达。利用定点突变对ChIFNα基因序列中4个糖基化位点进行缺失,SDS-PAGE分析N-糖基化对毕赤酵母表达ChIFNα的影响。结果:ChIFNα在GS115中获得了表达,在摇瓶发酵条件下,表达量为35.2 mg/L;构建了缺失1~4个糖基化位点的4个突变体,在GS115中实现了表达,分析表达结果显示,与未突变的ChIFNα对照相比,突变体的糖基化程度和表达量都有大幅度下降。结论:N-糖基化对于毕赤酵母表达ChIFNα具有重要影响,无糖基化的ChIFNα在毕赤酵母中表达量极低。     

氨基酸定点突变提高灵芝蛋白LZ-8热稳定性的研究

通过氨基酸定点突变技术提高灵芝免疫调节蛋白LZ-8的热稳定性。通过分子动力学模拟结合温度因子预测对LZ-8氨基酸突变位点进行理性设计,在毕赤酵母X33菌株内构建并表达LZ-8突变体蛋白,采用HeLa细胞生长抑制实验和差示量热扫描分析检测并比较了LZ-8突变前后生物学活性及热力学参数。结果显示,LZ-8 N-端α螺旋为理论预测的温度敏感区域,在该区域进行F8W和R9K氨基酸双位点突变,突变后的LZ-8热稳定性提高,相变温度Tm上升0.92℃,相转变焓值ΔH提高23.14 kJ/mol,但突变后LZ-8生物学活性基本不变,LZ-8和LZ-8突变体对HeLa细胞生长抑制的IC50值分别是2.238μg/mL和2.407μg/mL。通过理性设计氨基酸突变位点,获得了稳定性提高但生物学活性不变的灵芝免疫调节蛋白LZ-8的突变体。     

功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示*

目的:设计并构建新型冠状病毒(SARS-CoV-2)受体结合结构域(receptor binding domain,RBD)在毕赤酵母表面的展示体系,并对表面展示的RBD进行功能性评价,从而为以RBD为靶点的高通量药物筛选平台奠定基础。方法:将四种锚定分子与新冠病毒RBD融合,电转化至毕赤酵母中;通过细胞免疫荧光分析,筛选能够成功展示RBD的锚定系统;进一步分析其与血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)受体的亲和力,证明展示在细胞表面RBD分子的功能。结果:仅Sed1p锚定分子能够有效呈递RBD至毕赤酵母细胞表面,展示效率约为70%;亲和力分析结果表明,ACE2受体和表面展示RBD的亲和力(K_D = 30.42 nmol/L)与溶液中RBD的亲和力(K_D = 16.00 nmol/L)较为接近。结论:这一体系能够在毕赤酵母表面高效地展示具有生物学功能的RBD,可用于抗新冠病毒RBD药物的高通量筛选和评价。     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

基于重组毕赤酵母的fusaruside生物合成

目的:构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法:从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果:3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa; HPLC-MS显示重组酵母可以产生fusaruside。结论:与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

重组毕赤酵母产青霉素G酰化酶的分批发酵动力学研究

构建了重组毕赤酵母产青霉素G酰化酶的分批发酵动力学模型。实验考察了分批发酵过程中甘油消耗、甲醇浓度、菌体浓度、溶氧、补料时间对青霉素G酰化酶活力的影响。应用Matlab软件,对菌体生长、基质消耗和产物生成方程进行最优参数估算和非线性拟合,得到相应的动力学模型。模型的计算值与实验值能较好地拟合,表明所建模型能较好反映重组毕赤酵母产青霉素G酰化酶的分批发酵过程。     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross talk

Dopamine cellular signaling via the D(1) receptor (D(1)R) involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D(1)R-mediated inhibition of NADPH oxidase activity involves cross talk between PKA and a specific PKC isoform(s). In HEK-293 cells heterologously expressing human D(1)R (HEK-hD(1)), fenoldopam, a D(1)R agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D(1)R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two PKA inhibitors, H89 (10μmol/L; 88±8.1%) and Rp-cAMP (10μmol/L; 97.7±6.7%), and two PKC inhibitors, bisindolylmaleimide I (1μmol/L; 94±6%) and staurosporine (10nmol/L; 93±8%), which by themselves had no effect (n=4-8/group). The inhibitory effect of PMA (1μmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%; n=5 or 6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθ(S676), an effect that was also blocked by H89. Fenoldopam (1μmol/L) also increased PKCθ(S676) in HEK-hD(1) and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross talk between PKA and PKCθ plays an important role in the D(1)R-mediated negative regulation of NADPH oxidase activity in human kidney cells.     

Cloning of the nitrile hydratase gene from Nocardia sp. in Escherichia coli and Pichia pastoris and its functional expression using site-directed mutagenesis

To obtain a recombinant Rhodococcus or Nocardia with not only higher enzymatic activity but also better operational stability and product-tolerance ability for bioconversion of acrylamide from acrylonitrile, an active and stable expression system of nitrile hydratase (NHase) was tried to construct as the technical platform of genetic manipulations. Two NHase genes, NHBA and NHBAX, from Nocardia YS-2002 were successfully cloned, based on bioinformatics design of PCR primers, and inserted into plasmid pUC18 and pET32a, respectively. Then, two recombinant Escherichia coli strains, JM105 (pUC18-NHBA) and BL21 (DE3) (pET32a-NHBAX) were constructed and their expressions of NHase were focused. The induction results showed that there was either no NHase activity in JM105 (pUC18-NHBA), or as low as 0.04 U (1 U=1 μmol acrylamide min⁻¹ mg⁻¹ dry cell) in BL21 (DE3) (pET32a-NHBAX). SDS-PAGE results showed that the -subunit of NHBA and NHBAX could not be efficiently expressed in both recombinant E. coli strains. The novel Pichia pastoris system was also applied to express NHase, but the expression level remained quite low (0.5–0.6 U) and the protein was unstable. For solving this problem, a possible genetic strategy, site-directed mutagenesis of the -subunit of the NHase was carried out. After the successful mutagenesis of the original rare start codon gtg into atg, a new recombinant strain, E. coli XL1-Blue (pUC18-NHBA^M), was screened and the NHase activity stably reached as high as 51 U under the same induction conditions.