脱毛蛋白酶的纯化及其基因的克隆与表达研究
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摘要
短小芽孢杆菌(Bacillus pumilus)UN-42-C-31是一株从成都市生活垃圾中分离，经过多次反复诱变后得到的碱性蛋白酶产生菌。摇瓶和100L小型发酵实验结果表明，其发酵液中含有的碱性蛋白酶具有很好的脱毛效果，且对皮革胶原没有损伤。
对该菌株500L大规模发酵的蛋白酶活性检测发现，接种16h后开始产生蛋白酶，在28h达到高峰(4200U／mL)。对发酵液进行的硫酸铵饱和沉淀实验发现，在20-70％的硫酸铵饱和度范围内可以得到绝大部分(96％)的蛋白酶。
对发酵液进行SDS-PAGE和蛋白酶的活性染色分析表明，发酵液中的蛋白质种类不多，且只含有一种碱性蛋白酶。该发酵液所含蛋白酶活性能够被丝氨酸蛋白酶抑制剂PMSF，DFP完全抑制。该发酵液对牛皮，羊皮和猪皮均有良好的脱毛效果。
通过CM-sepharose Fast Flow离子交换层析，DEAE-sepharose Fast Flow离子交换层析，Sephacryl S-100，Sephacryl S-200凝胶过滤层析，疏水层析等纯化步骤对短小芽孢杆菌发酵液中的碱性蛋白酶进行了纯化。纯化产物通过SDS-PAGE电泳检查，已经达到了电泳纯标准。与纯化前的初酶液相比，酶的比活力提高了38.65倍，达到17530U／mg，但其回收率仅为0.88％。脱毛实验证明，纯化的蛋白酶能够单独完成整个脱毛过程。因此，该酶是一种脱毛碱性蛋白酶，命名为DHAP(Dehairing alkaline protease)。
四川大学博士学位论文
DHA卫的分子量约为3200oD，等电点为9.0，最适反应pH为10，最适反
应温度为58℃。该蛋白酶活性能够被丝氨酸蛋白酶的专一性抑制剂PMSF和
DFP完全抑制，同时也能被EGTA，EDTA等金属蛋白酶抑制剂所部分抑制。
此结果表明，DHAP为一种丝氨酸蛋白酶，其酶活性的发挥需要某些金属离子
的参与，如Na+，c扩+，M扩+等金属离子能提高DHAp的活性，但c了+与z矛十
能抑制DHAP的部分活性。
将纯化的DHAP进行转膜，测定了N一末端的加个氨基酸残基的序列，其
结果是:AQTvPYGIPQIKAP/汉HAQG。该序列与来自其它芽抱杆菌属菌株的
丝氨酸蛋白酶的N一末端氨基酸序列有较高的同源性，并和另一株短小芽抱杆菌
的丝氨酸蛋白酶N一末端氨基酸序列完全一致。
在此基础上，设计了用于扩增全长基因片段的引物，从短小芽抱杆菌的总
DNA中扩增得到了相应的片段。将扩增的全长基因片段用T载体进行克隆，然
后测定了全序列。该片段全长1 588bP，包括一个1 152bP的编码区序列，编码全
长为383个氨基酸残基的蛋白质。根据软件分析与同源性比较推测，该蛋白质
含有由29个氨基酸残基组成的信号肤，79个氨基酸残基组成的前肤。成熟蛋白
酶全长275个氨基酸残基，其中，D32，H64，和52213个氨基酸残基为催化活
性中心。
将克隆到的碱性蛋白酶基因(命名为Ap)编码的成熟蛋白N一端序列与纯
化的脱毛蛋白酶N一端序列进行比较，二者完全相同。同源性分析表明，该基因
序列与已报道的另一短小芽抱杆菌菌株的碱性蛋白酶基因序列有97%的同源
性，氨基酸序列同源性高达99.2%。但与其它丝氨酸蛋白酶基因相比，则同源
性低于60%。该蛋白酶可以认为是枯草杆菌蛋白酶家族(Subtilisin)的新成员。
将该蛋白酶基因插入大肠杆菌(EscheriaChia coli)表达载体pET-巧b，在
大肠杆菌中进行了表达。SDS一PAGE显示出相应的蛋白质带，含量约为总蛋白
的30%。表达的蛋白质形成包涵体，但经变性和复性后，未能表现出蛋白酶活
性。
将来自短小芽抱杆菌UN一31一c礴2的基因启动子(Bp53片段)和脱毛蛋白
酶全基因(AP)进行融合，然后将重组基因(命名为BpAp)插入到大肠杆菌-
枯草杆菌穿梭质粒载体psUGV4中，构建成表达质粒psu一BPAP。当把PsU一BpAp
转入到枯草杆菌WB600后，所得转化子能够在牛奶平板上产生透明的水解圈，
四川大学博士学位论文
在其发酵液中能够检测到蛋白酶活性，SDS一PAGE分析也能清晰的显示出相应
的蛋白质带，这些结果都表明脱毛蛋白酶在枯草杆菌中得到表达。转化子发酵
液也具有脱毛效果。
上述的研究结果表明，纯化后的短小芽抱杆菌DH妙和在枯草杆菌WB600
表达的DHAP都具有良好的脱毛活性，这些结果都说明了脱毛过程仅需一种蛋
白酶即可完成。
Bacillus pumilus UN-31-C-42 was obtained by mutations of strain BA(06), which is an alkaline protease producer and separated from life waste in ChengDu. The results of bith shaking and 100L fermentation experiments showed that the fermentation liquid has high dehairing and low collagen-degradation activity, indicating that the protease produced by this strain might be ideal enzyme agent for leather industry.
When this strain was fermented, it produced alkaline protease after inoculation of 16h and the enzyme activity got the peak at 28h with 4200U/mL. Ammonium sulfate precipitation experiment showed that about 96% of alkaline protease was recovered in the 20-70% concentration. Although SDS-PAGE displayed that there were several protein bands in the sample after 16h fermentation, enzymatic active stain demonstrated that there was only one band during the whole fermentation period, indicating that there is only one kind alkaline protease in the fermentation liquid. The enzyme activity in fermentation liquid could be inhibited by PMSF and DFP. The fermentation liquor also showed good dehairing activity.
The alkaline protease (named DHAP, dehairing alkaline protease) in the fermentation liquid was purified with hydrophobic interaction chromatography, ion exchange and gel filtration. The final yield was about 0.88% and the specific enzyme activity was increased 39-fold, with 17530 U/mg. The purified protease also displayed dehairing activity. In other words, DHAP can accomplish the whole process of dehairing by itself and without any other protease' s cooperation.
DHAP had a pI of 9.0 and a-molecular weight of approximate 32,000 Dal ton. The protease has the maximal activity at pHIO and 55癈. The enzyme activity could be completely inhibited by PMSF and DFP, and partially inhibited by pepstatin A, EDTA and EGTA. Leupeptin, Benzamidine and hydrochloride Aprotin had no influence on the DHAP activity. Ca2*, Mg2+ and Na* could slightly increase, but Cu2+ and Zn2+ slightly inhibited protease activity. All these results demonstrated that DHAP is a serine protease.
The first 20 amino acid residues of the purified DHAP were determined and the sequence is AQTVPYGIPQIKAPAVHAQG. The homologous analysis showed that the N-terminal sequence of DHAP is identical to that of serine protease from another B. pumilus strain TYO-67 and has high homology with those from other Bacillus species.
Based on the homologous analysis, three pairs of primers were designed and synthesized. Three fragments, containing intact gene, whole and mature protease coding region ware amplified from total DNA of B. pumilus UN-31-O42 by polymerase chain reaction, respectively. After cloned into T-vector, the sequence of intact gene fragment with the size of 1.6 kb, was determined. The gene contains an open reading frame of 1149 nucleotides (Named Ap gene, encoding 383 amino acids), which contains a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of protease.
Comparison of the entire derived peptide sequence with other serine protease revealed significant homology, especially with a reported gene from another B. pumilus strain TYO-67 (99% homology). The N-terminal amino acid sequence of deduced mature protease is identical to that of the purified alkaline protease. The sequence around these residues revealed that AP was a new member of the subtilisin family.
The gene was expressed in Escherichia coli after cloned into pET-15b. SDS-PAGE showed the expressed product clearly, but no protease activity was detected.
In order to express alkaline protease gene (AP gene) in Bacillus subtil is, the recombinant expression plasmid was constructed. This plasmid contains a promoter Bp53, also from B. pumilus UN-31-C-42, AP gene and the shuttle vector pSUGV4. After introduced into B. subtilis WB600, the transformants displayed the hydrolyzed zone on milk plate. The fermentation liquid of the transformant
对该菌株500L大规模发酵的蛋白酶活性检测发现，接种16h后开始产生蛋白酶，在28h达到高峰(4200U／mL)。对发酵液进行的硫酸铵饱和沉淀实验发现，在20-70％的硫酸铵饱和度范围内可以得到绝大部分(96％)的蛋白酶。
对发酵液进行SDS-PAGE和蛋白酶的活性染色分析表明，发酵液中的蛋白质种类不多，且只含有一种碱性蛋白酶。该发酵液所含蛋白酶活性能够被丝氨酸蛋白酶抑制剂PMSF，DFP完全抑制。该发酵液对牛皮，羊皮和猪皮均有良好的脱毛效果。
通过CM-sepharose Fast Flow离子交换层析，DEAE-sepharose Fast Flow离子交换层析，Sephacryl S-100，Sephacryl S-200凝胶过滤层析，疏水层析等纯化步骤对短小芽孢杆菌发酵液中的碱性蛋白酶进行了纯化。纯化产物通过SDS-PAGE电泳检查，已经达到了电泳纯标准。与纯化前的初酶液相比，酶的比活力提高了38.65倍，达到17530U／mg，但其回收率仅为0.88％。脱毛实验证明，纯化的蛋白酶能够单独完成整个脱毛过程。因此，该酶是一种脱毛碱性蛋白酶，命名为DHAP(Dehairing alkaline protease)。
四川大学博士学位论文
DHA卫的分子量约为3200oD，等电点为9.0，最适反应pH为10，最适反
应温度为58℃。该蛋白酶活性能够被丝氨酸蛋白酶的专一性抑制剂PMSF和
DFP完全抑制，同时也能被EGTA，EDTA等金属蛋白酶抑制剂所部分抑制。
此结果表明，DHAP为一种丝氨酸蛋白酶，其酶活性的发挥需要某些金属离子
的参与，如Na+，c扩+，M扩+等金属离子能提高DHAp的活性，但c了+与z矛十
能抑制DHAP的部分活性。
将纯化的DHAP进行转膜，测定了N一末端的加个氨基酸残基的序列，其
结果是:AQTvPYGIPQIKAP/汉HAQG。该序列与来自其它芽抱杆菌属菌株的
丝氨酸蛋白酶的N一末端氨基酸序列有较高的同源性，并和另一株短小芽抱杆菌
的丝氨酸蛋白酶N一末端氨基酸序列完全一致。
在此基础上，设计了用于扩增全长基因片段的引物，从短小芽抱杆菌的总
DNA中扩增得到了相应的片段。将扩增的全长基因片段用T载体进行克隆，然
后测定了全序列。该片段全长1 588bP，包括一个1 152bP的编码区序列，编码全
长为383个氨基酸残基的蛋白质。根据软件分析与同源性比较推测，该蛋白质
含有由29个氨基酸残基组成的信号肤，79个氨基酸残基组成的前肤。成熟蛋白
酶全长275个氨基酸残基，其中，D32，H64，和52213个氨基酸残基为催化活
性中心。
将克隆到的碱性蛋白酶基因(命名为Ap)编码的成熟蛋白N一端序列与纯
化的脱毛蛋白酶N一端序列进行比较，二者完全相同。同源性分析表明，该基因
序列与已报道的另一短小芽抱杆菌菌株的碱性蛋白酶基因序列有97%的同源
性，氨基酸序列同源性高达99.2%。但与其它丝氨酸蛋白酶基因相比，则同源
性低于60%。该蛋白酶可以认为是枯草杆菌蛋白酶家族(Subtilisin)的新成员。
将该蛋白酶基因插入大肠杆菌(EscheriaChia coli)表达载体pET-巧b，在
大肠杆菌中进行了表达。SDS一PAGE显示出相应的蛋白质带，含量约为总蛋白
的30%。表达的蛋白质形成包涵体，但经变性和复性后，未能表现出蛋白酶活
性。
将来自短小芽抱杆菌UN一31一c礴2的基因启动子(Bp53片段)和脱毛蛋白
酶全基因(AP)进行融合，然后将重组基因(命名为BpAp)插入到大肠杆菌-
枯草杆菌穿梭质粒载体psUGV4中，构建成表达质粒psu一BPAP。当把PsU一BpAp
转入到枯草杆菌WB600后，所得转化子能够在牛奶平板上产生透明的水解圈，
四川大学博士学位论文
在其发酵液中能够检测到蛋白酶活性，SDS一PAGE分析也能清晰的显示出相应
的蛋白质带，这些结果都表明脱毛蛋白酶在枯草杆菌中得到表达。转化子发酵
液也具有脱毛效果。
上述的研究结果表明，纯化后的短小芽抱杆菌DH妙和在枯草杆菌WB600
表达的DHAP都具有良好的脱毛活性，这些结果都说明了脱毛过程仅需一种蛋
白酶即可完成。
Bacillus pumilus UN-31-C-42 was obtained by mutations of strain BA(06), which is an alkaline protease producer and separated from life waste in ChengDu. The results of bith shaking and 100L fermentation experiments showed that the fermentation liquid has high dehairing and low collagen-degradation activity, indicating that the protease produced by this strain might be ideal enzyme agent for leather industry.
When this strain was fermented, it produced alkaline protease after inoculation of 16h and the enzyme activity got the peak at 28h with 4200U/mL. Ammonium sulfate precipitation experiment showed that about 96% of alkaline protease was recovered in the 20-70% concentration. Although SDS-PAGE displayed that there were several protein bands in the sample after 16h fermentation, enzymatic active stain demonstrated that there was only one band during the whole fermentation period, indicating that there is only one kind alkaline protease in the fermentation liquid. The enzyme activity in fermentation liquid could be inhibited by PMSF and DFP. The fermentation liquor also showed good dehairing activity.
The alkaline protease (named DHAP, dehairing alkaline protease) in the fermentation liquid was purified with hydrophobic interaction chromatography, ion exchange and gel filtration. The final yield was about 0.88% and the specific enzyme activity was increased 39-fold, with 17530 U/mg. The purified protease also displayed dehairing activity. In other words, DHAP can accomplish the whole process of dehairing by itself and without any other protease' s cooperation.
DHAP had a pI of 9.0 and a-molecular weight of approximate 32,000 Dal ton. The protease has the maximal activity at pHIO and 55癈. The enzyme activity could be completely inhibited by PMSF and DFP, and partially inhibited by pepstatin A, EDTA and EGTA. Leupeptin, Benzamidine and hydrochloride Aprotin had no influence on the DHAP activity. Ca2*, Mg2+ and Na* could slightly increase, but Cu2+ and Zn2+ slightly inhibited protease activity. All these results demonstrated that DHAP is a serine protease.
The first 20 amino acid residues of the purified DHAP were determined and the sequence is AQTVPYGIPQIKAPAVHAQG. The homologous analysis showed that the N-terminal sequence of DHAP is identical to that of serine protease from another B. pumilus strain TYO-67 and has high homology with those from other Bacillus species.
Based on the homologous analysis, three pairs of primers were designed and synthesized. Three fragments, containing intact gene, whole and mature protease coding region ware amplified from total DNA of B. pumilus UN-31-O42 by polymerase chain reaction, respectively. After cloned into T-vector, the sequence of intact gene fragment with the size of 1.6 kb, was determined. The gene contains an open reading frame of 1149 nucleotides (Named Ap gene, encoding 383 amino acids), which contains a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D32, H64, and S221, were identical with 3 essential amino acids in the catalytic center of protease.
Comparison of the entire derived peptide sequence with other serine protease revealed significant homology, especially with a reported gene from another B. pumilus strain TYO-67 (99% homology). The N-terminal amino acid sequence of deduced mature protease is identical to that of the purified alkaline protease. The sequence around these residues revealed that AP was a new member of the subtilisin family.
The gene was expressed in Escherichia coli after cloned into pET-15b. SDS-PAGE showed the expressed product clearly, but no protease activity was detected.
In order to express alkaline protease gene (AP gene) in Bacillus subtil is, the recombinant expression plasmid was constructed. This plasmid contains a promoter Bp53, also from B. pumilus UN-31-C-42, AP gene and the shuttle vector pSUGV4. After introduced into B. subtilis WB600, the transformants displayed the hydrolyzed zone on milk plate. The fermentation liquid of the transformant
引文
1．奥斯泊，F.,R．布伦特，RE．金斯顿，DD．穆尔，JG．塞德曼，JA．史密斯，K．斯特拉尔，1999，精编分子生物学实验指南(Short Protocolsin MoLecular Biology)，科学出版社
2．高忠柏，1997，中国皮革必须走可持续发展之路，中国皮革，26(2)：21—23
3．何塞，卢芩贝格，(黄风祝译)，1999，自然不可改良，生活．读者．新知三联书店
4．刘白玲，何先祺，张义正，1997，枯草杆菌中性蛋白酶基因在大肠杆菌中的表达，生物工程学报，13(3)：304-308
5．刘白玲，1992，枯草杆菌蛋白酶基因的分离鉴定及表达的研究，成都科技大学博士论文
6．刘成君，1998，枯草杆菌HX-318菌株特性研究和皮革脱毛蛋白酶基因的克隆，四川大学博士后研究工作报告
7．林斌，王贤舜，郑屹，邓立，李冰，1998，地衣芽孢杆菌2709碱性蛋白酶地分离纯化和性质鉴定，生物化学和生物物理学报，25(3)：287—292
8．卢戈，1991，胶原的分子类型、结构分级及与细胞外基质成分的关系，生物化学与生物物理进展，18(4)：275-280
9．马歇克，DR.,JT．门永布，RR．格斯克，MW．努特，WA．布伦南，SH．林，1999，蛋白质纯化与鉴定实验指南(Strategies for Protein purification and Characterization)，科学出版社
10．沈同，王镜岩(主编)，1990，生物化学，p282-289．高等学校出版社
11．田美，中国皮革工业现状分析及前景展望，1997，中国皮革，26(1)：7—13
12．汤克勇，王芳，苏智健，宋丽英，张铭让，2000，皮革工业必须走可持续发展之路，中国皮革，29(7)：19—22
13．熊振平，1989，酶工程，化学工业出版社
14．张鸣让，林炜，2001，绿色化学和技术与皮革工业的可持续发展，中国皮革，30(1)：5—12
15．张树政，王修恒，1988，工业微生物成就，科学出版社，P79-81
16．张淑华，世界皮革工业形势和我们的对策，1995，皮革化工，3：10—16
17．张义正，刘德明，刘彦，程昌凤，黄庆，脱毛蛋白酶、其制造方法、使用方法和产生此蛋白酶的微生物，2002，中国专利：00120641.9
18. Bach H., D.Errampalli, KT.Leung, H.Lee, A.Hartmann, JT.Trevors and JC.Muneh, 1999, Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization., Appl. Environ. Microbiol., 65(7): 3226-3228
19. Cocheo V., 1990, Environmental impact of tanning industry., Med. Lav., 81(3): 230-241.
20. Ephrati-Elizur E., PR. Strinivasan and S. Zamenhof., 1961, Genetic analysis by means of transformation of histidine linkage groups in Bacillus subtilus., Proc. Nati. Acad. Sci. USA. 47:56-63
21. Feairheller SH., MM.Taylor, W.Windus, EM.Filachione and J.Naghski., 1972, Recovery and analyses of hair proteins from tannery unhaidng wastes., J. Agdc. Food. Chem., 20(3): 668-670.
22. Jonczyk W. and K. Studniarski, 1986, Enzyme unhairing of pigskins., Journal of Society of Leather Technologists and Chemists, 72:83-88
23. Leighton TJ., RH. Doi and RA. Kelln., 1973, The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis., J. Mol. Biol., 76:103-122
24. Loperena L., MD.Ferrari, L.Belobrajdic, R.Weyrauch and H.Varela, 1994, Study of.Bacillus sp. culture conditions to promote production of unhairing proteases., Rev. Argent Microbiol., 26(3): 105-115.
25. Makinen KK. and PL.Makinen, 1987, Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67)., J. Biol. Chem., 262(26): 12488-12495
26. Made HR., EM. Brahim, G. Machhour, L. Catherine and LJ. Jean, 1994, Electrophoretie Detevtion of Cytoplasmic Serine Proteinases in Candida albicans, ExPerimental Mycology, 18:267-270
27.Mervyn L and EL.Smith, 1964, The biochemistry of vitamin B12 fermentation., Prog. Ind. Microbiol., 5:151-201
28.Ogura M., M.Kawata-Mukai, M.Itaya, K. Takio and T.Tanaka, 1994, Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis., J. Bacteriol., 176(18): 5673-5680
29.Patnaik PR., 2001, Penicillin fermentation: mechanisms and models for industrial-scale bioreactors., Crit. Rev. Microbiol., 27(1): 25-39.
30.Puvanakrishnan R. and SC.Dhar, 1986, Recent Advances in the Enzymatic Depilution of Hides and Skins., Leather Science, 33:177-191
31.Sloma A., GA.Rufo, KA.Theriault, M.Dwyer, SW.Wilson and J.Pero, 1991, Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis., J. Bacteriol., 173(21): 6889-6895.
32.Taylor M., D.G.Bailey and S.H.Feairheller, 1987, A review of the use of enzymes in the Tannery., J.A.L.C.A., 82:153-165
33.Yates JR., 1969, Studies in depilation.V.Investigation of the relative depilatory efficiency of a number of commericially available enzyme system, J.A.L.C.A. 63:464-479
2．高忠柏，1997，中国皮革必须走可持续发展之路，中国皮革，26(2)：21—23
3．何塞，卢芩贝格，(黄风祝译)，1999，自然不可改良，生活．读者．新知三联书店
4．刘白玲，何先祺，张义正，1997，枯草杆菌中性蛋白酶基因在大肠杆菌中的表达，生物工程学报，13(3)：304-308
5．刘白玲，1992，枯草杆菌蛋白酶基因的分离鉴定及表达的研究，成都科技大学博士论文
6．刘成君，1998，枯草杆菌HX-318菌株特性研究和皮革脱毛蛋白酶基因的克隆，四川大学博士后研究工作报告
7．林斌，王贤舜，郑屹，邓立，李冰，1998，地衣芽孢杆菌2709碱性蛋白酶地分离纯化和性质鉴定，生物化学和生物物理学报，25(3)：287—292
8．卢戈，1991，胶原的分子类型、结构分级及与细胞外基质成分的关系，生物化学与生物物理进展，18(4)：275-280
9．马歇克，DR.,JT．门永布，RR．格斯克，MW．努特，WA．布伦南，SH．林，1999，蛋白质纯化与鉴定实验指南(Strategies for Protein purification and Characterization)，科学出版社
10．沈同，王镜岩(主编)，1990，生物化学，p282-289．高等学校出版社
11．田美，中国皮革工业现状分析及前景展望，1997，中国皮革，26(1)：7—13
12．汤克勇，王芳，苏智健，宋丽英，张铭让，2000，皮革工业必须走可持续发展之路，中国皮革，29(7)：19—22
13．熊振平，1989，酶工程，化学工业出版社
14．张鸣让，林炜，2001，绿色化学和技术与皮革工业的可持续发展，中国皮革，30(1)：5—12
15．张树政，王修恒，1988，工业微生物成就，科学出版社，P79-81
16．张淑华，世界皮革工业形势和我们的对策，1995，皮革化工，3：10—16
17．张义正，刘德明，刘彦，程昌凤，黄庆，脱毛蛋白酶、其制造方法、使用方法和产生此蛋白酶的微生物，2002，中国专利：00120641.9
18. Bach H., D.Errampalli, KT.Leung, H.Lee, A.Hartmann, JT.Trevors and JC.Muneh, 1999, Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization., Appl. Environ. Microbiol., 65(7): 3226-3228
19. Cocheo V., 1990, Environmental impact of tanning industry., Med. Lav., 81(3): 230-241.
20. Ephrati-Elizur E., PR. Strinivasan and S. Zamenhof., 1961, Genetic analysis by means of transformation of histidine linkage groups in Bacillus subtilus., Proc. Nati. Acad. Sci. USA. 47:56-63
21. Feairheller SH., MM.Taylor, W.Windus, EM.Filachione and J.Naghski., 1972, Recovery and analyses of hair proteins from tannery unhaidng wastes., J. Agdc. Food. Chem., 20(3): 668-670.
22. Jonczyk W. and K. Studniarski, 1986, Enzyme unhairing of pigskins., Journal of Society of Leather Technologists and Chemists, 72:83-88
23. Leighton TJ., RH. Doi and RA. Kelln., 1973, The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis., J. Mol. Biol., 76:103-122
24. Loperena L., MD.Ferrari, L.Belobrajdic, R.Weyrauch and H.Varela, 1994, Study of.Bacillus sp. culture conditions to promote production of unhairing proteases., Rev. Argent Microbiol., 26(3): 105-115.
25. Makinen KK. and PL.Makinen, 1987, Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67)., J. Biol. Chem., 262(26): 12488-12495
26. Made HR., EM. Brahim, G. Machhour, L. Catherine and LJ. Jean, 1994, Electrophoretie Detevtion of Cytoplasmic Serine Proteinases in Candida albicans, ExPerimental Mycology, 18:267-270
27.Mervyn L and EL.Smith, 1964, The biochemistry of vitamin B12 fermentation., Prog. Ind. Microbiol., 5:151-201
28.Ogura M., M.Kawata-Mukai, M.Itaya, K. Takio and T.Tanaka, 1994, Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis., J. Bacteriol., 176(18): 5673-5680
29.Patnaik PR., 2001, Penicillin fermentation: mechanisms and models for industrial-scale bioreactors., Crit. Rev. Microbiol., 27(1): 25-39.
30.Puvanakrishnan R. and SC.Dhar, 1986, Recent Advances in the Enzymatic Depilution of Hides and Skins., Leather Science, 33:177-191
31.Sloma A., GA.Rufo, KA.Theriault, M.Dwyer, SW.Wilson and J.Pero, 1991, Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis., J. Bacteriol., 173(21): 6889-6895.
32.Taylor M., D.G.Bailey and S.H.Feairheller, 1987, A review of the use of enzymes in the Tannery., J.A.L.C.A., 82:153-165
33.Yates JR., 1969, Studies in depilation.V.Investigation of the relative depilatory efficiency of a number of commericially available enzyme system, J.A.L.C.A. 63:464-479