【作者】 柯野；

【导师】 谢明权；

【作者基本信息】 华南理工大学 ， 微生物学， 2013， 博士

【摘要】 霉菌是我国传统豆类发酵食品的主要生产菌种，有着悠久的应用历史。由于长期受到环境条件（高蛋白培养基）的驯化，这些霉菌具有分泌多种胞外蛋白酶的能力；这些胞外蛋白酶对植物蛋白具有高效的水解能力，特别对大豆蛋白而言，水解产物无苦味且水解程度高。目前，国内外学者对霉菌的研究主要集中于优化固体（或者液体）发酵条件来提高霉菌蛋白酶的产量或对酶学性质的研究；而对霉菌蛋白酶基因的克隆表达方面的研究鲜有报道。鉴于此，本课题对几种霉菌蛋白酶基因进行了克隆表达、定点突变和水解大豆分离蛋白等方面的研究。利用RACE和PCR技术从Actinomucor elegans、Rhizopus chinensis、Aspergillus niger和Aspergillus oryzae等几种霉菌中克隆获得3个新氨肽酶基因（GenBank登录号分别为HQ825158、JQ657815和JQ657814，下同）、2个新丝氨酸蛋白酶基因（GU356536和JF922913）；进一步获得4个丝氨酸蛋白酶基因（L19059、XM₀01391433、XM₀01824768和XM₀01820092）、5个酸性蛋白酶基因（XM₀01399818、XM₀01401056、D13894、AB090877和AB044079）和1个中性蛋白酶基因（AF099904）。本试验共克隆获得15个蛋白酶基因。根据生物信息学的理论和方法，对这些蛋白酶基因进行了较全面的预测和分析。结果表明，这些蛋白酶基因的密码子偏好性均不相同，并且编码的氨基酸残基长度也不一致。6种丝氨酸蛋白酶属于蛋白酶K家族，亲缘关系较近；以蛋白酶K晶体结构（PDBcode:1IC6A）作为模板进行同源建模分析可知：这些蛋白酶分子内均无二硫键，具有1个Ca2+结合位点；蛋白酶之间以及与蛋白酶K在底物结合区域、氢键、盐键和二硫键等方面均存在差异，这些差异可能是这些酶具有独特水解作用的原因。5种酸性蛋白酶属于酸性蛋白酶Asp家族，是胞外蛋白酶，亲缘关系较近；以Aspergillopepsin I晶体结构（PDB code:1IBQ B）作为模板进行同源建模可知：蛋白酶分子内均有1个二硫键，具有1个Zn2+结合位点。蛋白酶之间在活性位点的溶剂可及表面积、氢键、盐键、底物结合区域和ψ-loop结构等方面均存在差异，这些差异可能使这些蛋白酶具有独特的酶学性质。对中性蛋白酶和氨肽酶的功能位点、Motif结构和进化关系进行了分析；由于中性蛋白酶和氨肽酶未具有较为适合的同源建模模板，因此未能构建模拟出二级结构和三级结构的模型。分别将A. elegans、R. chinensis的Alp基因、A.niger的AlpI基因、A. oryzae的AlpII基因和A. elegansAmp基因克隆至表达载体pET-22b（+）上，转化至大肠杆菌BL21菌株中进行诱导表达；结果表明这些蛋白酶基因均被诱导表达出未有活性的重组蛋白酶。将蛋白酶基因克隆至表达载体pPIC9K上，电转整合至毕赤酵母Km71菌株的基因组上进行诱导表达。结果表明只有A.niger的AlpI基因、A.oryzae的AlpII基因和NpI基因能成功表达。进一步分别对3种重组蛋白酶进行了分离纯化和酶学性质研究。结果表明：重组黑曲霉AlpI在10L发酵罐中诱导表达时，发酵液的蛋白酶酶活达到4050U/mL，该酶最适反应pH值和温度分别为8.0-9.0和45℃，在pH为6.0-9.0和温度低于40℃时具有较好的热稳定性，PMSF完全抑制酶活，Ca2+、Mn2+和K+对酶具有促进作用，Zn2+、Fe2+和Mg2+都具有抑制作用。重组米曲霉AlpII在10L发酵罐中诱导表达时，发酵液的蛋白酶酶活达4100U/mL；该蛋白酶最适反应pH值和温度分别为8.5-9.5和50℃，在pH值为6.0-10.0和温度低于40℃时具有较好的稳定性；PMSF完全抑制酶活；Ca2+和Mg2+具有促进酶的作用，Zn2+、Fe2+和Mn2+具有轻微的抑制作用。重组米曲霉NpI在10L发酵罐中诱导表达时，发酵液的蛋白酶酶活达43101U/mL；该酶最适反应pH和温度分别为8.0和55℃，在pH为5.0-9.0和45℃以下具有较好的稳定性，EDTA强烈抑制酶活，Cu2+和Zn2+能显著的抑制酶活。为了提高重组蛋白酶的热稳定性利于工业的开发利用，采用定点突变技术来增加蛋白酶的二硫键。结果表明：重组米曲霉AlpII的突变酶G33C-S126C（将33位点和126位点氨基酸G和S突变为C）提高了热稳定性；但是突变酶I179C-T253C的热稳定性发生了降低；突变酶G33C-S126C-179C-T253C不能被表达。重组黑曲霉AlpI的3种突变酶（G34C-S127C、I180C-T254C和G34C-S127C-180C-T254C），均不能在毕赤酵母KM71菌株中成功表达。对米曲霉NpI的“HExxH¹9aa^E”Motif结构中的活性位点（H429、H433和E453）进行一系列的突变，结果表明该3个位点对NpI非常重要，进一步说明该酶属于锌蛋白酶家族。以大豆蛋白为底物，考察了重组蛋白酶对大豆分离蛋白的水解能力。结果表明重组米曲霉ApII最佳温度、pH值、水解时间和加酶量分别为45℃、10.0、1-1.5h和1000U/g，此时大豆分离蛋白水解度为15.8%。重组黑曲霉ApI最佳的温度、pH值、水解时间和加酶量分别为40℃、10.0、1h和1000U/g，此时的大豆分离蛋白水解度为15.6%。重组米曲霉NpI最佳的温度、pH值、水解时间和加酶量分别为45℃、8.0、1-1.5h和1500U/g，此时的大豆分离蛋白水解度为16.4%。更多还原

【Abstract】 Some fungi strains were the predominant strains used in the production of traditionalfermentation food of soybean in china. Because of a long time of acclimatization on the highprotein medium, these strains could synthesize and secrete several proteinases, which hadgood suitability to plant protein and showed high hydrolysis efficiency to plant protein,especially for soybean protein. At present, researches of both domestic and abroad werefoucused on improving yields of proteases by optimizing solid or liquid fermentation andenzymic properties, while neglected cloning and expression of protease genes from fungi. Forthis reason, our research focused on clone and expression of protease genes from severalfungi, site-directed mutagenesis, and hydrolysis efficiency to soybean protein.Three new aminopeptidase genes from fungi, such as Actinomucor elegans, Rhizopuschinensis and Mucor racemosus, etc. were achieved, and respectively submitted to GenBank（access number HQ825158, JQ657815and JQ657814, the below is same）, and two newserine protease genes （GU356536and JF922913）. Further, other protease genes were gainedby RT-PCR techniques, these genes were as follow: four serine protease genes （L19059,XM₀01391433, XM₀01824768and XM₀01820092）, five acid proteases genes（XM₀01399818, XM₀01401056, D13894, AB090877and AB044079）, and one neutralprotease genes （AF099904）. In a word, fifteen proteases genes were obtain in our research.These protease genes were predicted and analyzed by in detail based on thebioinformatics theory and technology. The results showed all protease genes, which haddifferent preference for codon usage, coded different length of amino acid residues. Six kindsof proteases belonged to Proteinase K family and exhibited the closest genetic relationshipeach other. Moreover, according to the six proteases structure modeled via homologymodeling with Proteinase K （PDB code:1IC6） as the crystal coordinate, it was found that sixproteases had no disulfide bonds and possessed a Ca2+binding site, and that the differences insubstrate-binding region, hydrogen bond, salt bridge and disulfide bond between proteaseswere probably responsible for the special hydrolysis ability.Five kinds of proteases belonged to acid protease Asp family, and were extracellularproteases, and exhibited the closest genetic relationship each other. Moreover, according to the five proteases structure modeled via homology modeling with Aspergillopepsin I （PDBcode:1IBQ B） as the crystal coordinate, it was found that five proteases respectively had adisulfide bond and possessed a Zn2+binding site. The differences in the solvent accessiblesurface area of active sites, hydrogen bond, salt bridge, substrate-binding region and ψ-loopstructure between five proteases were probably responsible for the special enzymaticcharacteristics.Active sites, motif structure and evolutionary relationship of the neutral protease and theaminopeptidase genes respectively were predicted and analyzed. Because these proteases hadno suitable homology models, the secondary structure and the tertiary structure were notpredicted.The fragments containing protease genes, which respectively were from A.elegans, R.chinensis, A.niger, A.oryzae and A.elegans, were cloned into pET-22b（+） expression vector,then were transformed into E. coli Bl21and expressed under the induction of IPTG. Theresults showed the expressed recombinant proteases were inactive proteases. These proteasegenes were cloned into the secreted expression pPIC9K vector, and the recombinant vectorswere transferred into Pachia pastoris KM71strain, then the recombinant KM71strains wereinduced using methyl hydrate. The results showed only three protease genes could beexpressed sucessfuly, and the three protease genes respectively was AlpI gene of A.niger,AlpII gene of A.oryzae and NpI gene of A.oryzae. Furtherly, the characters of the threerecombinant proteases separated and purified respectively were studied. The results ofrecombinant protease（rAlpI） of A.niger showed that （1） when the protease were induced toexpress in a10-L fermentor，the protease activity of the fermentation broth was up to4050U/mL;（2） the optimum pH value and temperature of the protease were respectively8.0-9.0and50℃，and the protease was stable at a pH value of6.0-9.0below40℃;（3） PMSFcould absolutely inhibite the activity of the protease, and the the activity of recombinantprotease was respectively activated by Ca2+, Mn2+and K+, and slightly inhibited by Zn2+、Fe2+and Mg2+.The results of recombinant protease（rAlpII） of A.oryzae showed that （1） when theprotease was induced to express in a10-L fermentor，the protease activity of the fermentationbroth was up to4100U/mL;（2） the optimum pH value and temperature of the protease were respectively8.5-9.5and50℃, and the protease was stable at a pH value of6.0to10.0below40℃;（3） PMSF could completely inhibite the activity of protease, and the activity ofrecombinant protease was respectively activated by Ca2+and Mg2+, and slightly inhibited byZn2+,Fe2+and Mn2+.The results of recombinant protease（rNpI） of A.oryzae showed （1） when the proteasewas induced to express in a10-L fermentor，the protease activity of the fermentation brothwas up to43101U/mL;（2）the optimum pH value and temperature of the protease wererespectively8.0and55℃, and the protease was stable at a pH value of5.0to9.0below45℃;（2）EDTA could completely inhibited the activity of protease, and the activity of recombinantprotease was inhibited by Cu2+and Zn2+.In order to improve the thermal stability of the recombinant proteases for industrialexploitation and utilization, the site directed mutagenesis have been used to add the disulfidebonds into the recombinant proteases. The results showed that the thermal stability of themutant enzyme AlpII G33C-S126C of A.oryzae, which indicated that amino acids residuals ofsite33and site126were mutated into C, were improved, while the mutant enzymeI179C-T253C was reduced, and the mutant enzyme G33C-S126C-179C-T253C could not beexpressed in P. pastoris. The three mutant enzymes of A.niger, G34C-S127C, I180C-T254Cand G34C-S127C-180C-T254C, could not be expressed in P. pastoris, too. A series ofmutations to “HExxH¹9aa^E” Motif of the rNpI of A.oryzae were constructed, the resultsshowed the three active sites were important to the the rNpI, and further was confirmed theprotease NpI as a gluzincin.Using isolated soybean proteins as substrate, the hydrolysis efficiency of the threerecombinant proteinases was studied. The results indicated （1） that the rApII of A.oryzae hadthe highest hydrolysis degree （15.8%） at45℃, pH10.0,1-1.5h and1000U/g;（2） the rApI ofA.niger had the highest hydrolysis degree （15.6%） at40℃, pH10.0,1h and1000U/g;（3） therNpI of A.oryzae had the highest hydrolysis degree （16.4%） at45℃, pH8.0,1-1.5h and1500U/g.更多还原