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INHIBITORY PROPERTY CHARACTERIZATION AND REACTIVE SITE EXPLORATION OF THE ARROWHEAD proteinase INHIBITOR

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Science China Chemistry 1991年第7期34卷 832-839页

作者：杨慧玲王丽秀朱德煦戚正武 Shanghai Institute of Biochemistry Academia Sinica Shanghai 200031 PRC

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymo-trypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymo-trypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the structural characteristics of inhibitors A and B, it was predicated that the two reactive sites should be located in the positions Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), respectively. In inhibitor A, there exists another hydrophobic residue involved in inhibiting chymotrypsin. This residue might be situated in the reactive region composed of the Arg reactive site.

关键词： Ser proteinase inhibitor arrowhead chemical modification.

Transgenic Tobacco Plants by Cotransformation With proteinase Inhibitor Ⅱ and δ-endotoxin Genes

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Chinese Science Bulletin 1993年第18期38卷 1561-1566页

作者：华学军陈晓邦马万超范云六 Molecular Biology Laboratory Biotechnology Research Centre Chinese Academy of Agricultural Sciences Beijing 100080 PRC

In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the insect resistance to B. t δ-endotoxin produced by transgenic plants has been paid attention to. To combine one B. t δ-endotoxin gene with an incompatible B. t δ-endotoxin gene or with other genes such as proteinase inhibitor gene might alleviate the insect resistance problem. In this note, we study the

关键词： proteinase inhibitor gene B. t δ-endotoxin gene cotransformation transgenic plants gene expression.

NMR studies of the interaction between Bacillis subtilis neutral proteinase and inorganic metal compounds

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Chinese Science Bulletin 2000年第19期45卷 1764-1766页

作者： ZHENG Xuefang HU Jiehan XU Yongting CHENG Guobao GAI Hongwei Deparlmenl of Chemistry and Chemical Engineering Dalian UniversityDalian 116622China Dalian Institute of Chemical Physics Chinese Academy of SciencesDalian 116023China Experimental Centre of Liaoning Normal University Dalian 116021China

The interaction between Bacillis subtilis neutral proteinase (***) and inorganic metal compounds (CoCI2, NiCI2) was investigated by 1H NMR spectroscopy. It has been shown that the Zn(Ⅱ) ion in the active center of the native enzyme may directly interact with external CoCI2 and NiCI2, producing Co(Ⅱ)- and Ni(Ⅱ)-substituted derivatives, and their 1H NMR spectra were obtained for the first time. From the 1H NMR spectra, the coordinated structure of the active center in the native enzyme was described.

关键词： Bacillis subtilis neutral proteinase CoCl2 NiCl2 interaction NMR.

Isolation of tomato proteinase inhibitor Ⅱ gene and the function of its intron

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Chinese Science Bulletin 2002年第10期47卷 830-834页

作者： Xie, XZ Wu, NH Chinese Acad Sci Inst Dev Biol Beijing 100080 Peoples R China

The genomic DNA sequence of tomato pro-teinase inhibitor II gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucieotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.

关键词： tomato proteinase inhibitor II gene intron enhancer.

Biocatalytic direct asymmetric aldol reaction using proteinase from Aspergillus melleus

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Science China Chemistry 2013年第7期56卷 939-944页

作者： YUAN Yi GUAN Zhi HE YanHong School of Chemistry and Chemical Engineering Southwest University

The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.

关键词： enzyme catalysis aldol reaction proteinase promiscuity asymmetric synthesis

A proteinase from Mung Bean Sprouts That Inactivaties Soybean Trypsin Inhibitor

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Acta Botanica Sinica 2001年第11期43卷 1150-1153页

作者：陈中杨晓泉赵谋明华南理工大学食品与生物工程学院广州510641

By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.

关键词： proteinase inactivate soybean trypsin inhibitor

A new method of research on molecular evolution of proteinase superfamily

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Chinese Science Bulletin 2001年第7期46卷 578-582页

作者： SUN Zhirong JI Xinglai HU Shengmin WANG Yu HE Fuchu Department of Biology Tsinghua UniversityBeijing 100084China Radiate Medicine Institute Academy of Military Medical SciencesBeijing 100850China

The molecular evolutionary tree, also known as a phylogenetic tree, of the serine proteinase superfamily was constructed by means of structural alignment. Three-dimensional structures of proteins were aligned by the SSAP program of Orengo and Taylor to obtain evolutionary distances. The resulting evolutionary tree provides a topology graph that can reflect the evolution of structure and function of homology proteinase. Moreover, study on evolution of the serine proteinase superfamily can lead to better understanding of the relationship and evolutionary difference among proteins of the superfamily, and is of significance to protein engineering, molecular design and protein structure prediction. Structure alignment is one of the useful methods of research on molecular evolution of protein.

关键词： molecular evolution SSAP algorithm structure alignment evolutionary tree serine proteinase superfamily.

Molecular Cloning of a Thiol proteinase Inhibitor Gene and Its Expression in ***

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High Technology Letters 1996年第2期2卷 89-94页

作者：周兆斓 Institute of Genetics Academia SinicaBeijing 100101P.R.China

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in *** the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins *** papain-inhibitory activity of the expressed OC was further confirmed.

关键词： Rice cDNA library Thiol proteinase inhibitor Insect-resistant gene Sequence analysis High level expression

On the Activities of Pancreatic Proteases and Alpha-1 proteinase Inhibitor in Meat-Type Chicken

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Open Journal of Animal Sciences 2017年第3期7卷 289-296页

作者： Vladimir G. Vertiprakhov Alena A. Grozina Ivan A. Egorov Tatiana N. Lenkova Vardges A. Manukyan Tatiana A. Egorova Federal Scientific Center “All-Russian Research and Technological Poultry Institute” of Russian Academy of Sciences Moscow Russia

The study was aimed at the evaluation of the effects of breed, age, different digestion stimulators, and dietary crude protein (CP) level on the activities of proteolytic enzymes in pancreatic tissue and duodenal chymus (in vivo), serum trypsin and α1-proteinase inhibitor (A1PI) concentrations in meat-type chicks. The study of age dynamics of trypsin and A1PI concentrations was performed on the chicks of hybrid cross “Smena-8”and two parental lines (Plymouth Rock and Cornish) fed standard commercial corn-wheat-SBM diets. Twenty birds per breed were euthanized at 1, 7, 14, 21, 28 and 35 days of age to obtain blood samples and pancreatic homogenate. Experiments on the effects of different digestion promotors (probiotic, acidifier, phytobiotic, enzymatic preparation) and different CP levels (finisher diet, CP 20%, vs. ground corn, CP 8.5%) were performed on 12 hybrid chicks with fistulated duodenum from 14 to 50 days of age. The following conclusions were made: 1) At 1 day of age high proteolytic activity in pancreatic tissue and maximal serum concentrations of trypsin and A1PI were found in both hybrid and parental lines. Since 7 to 35 days of age A1PI concentration was nearly constant, serum trypsin concentration decreased while proteolytic activity in pancreatic tissue exhibited undulate increase;2) Proteolytic activity in pancreatic tissue was higher in hybrids compared to the parental lines from 7 to 35 days of age (p 0.05);3) Supplementation of diet with exogenous enzymes stimulated the digestion due to the increase in protease activity in duodenal chymus by 9.1% compared to unsupplemented control (p 0.05);4) Proteolytic activity in duodenal chymus significantly responded to the substitution of ground corn for the complete diet by 2-fold decrease while serum trypsin concentration responded by 2.5-fold increase (p 0.001). This fact can indicate that physiological functions of digestive proteases are not confined to the digestive processes.

关键词： Chicks Pancreas Trypsin Alpha-1 proteinase Inhibitor (Antitrypsin) Serum Duodenal Fluid